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CN116064273B - Helicobacter pylori standard strain JMF0 and FMA6 containing specific molecular targets, detection and application thereof - Google Patents

️Tue Aug 22 2023

Helicobacter pylori standard strain JMF0 and FMA6 containing specific molecular targets, detection and application thereof Download PDF

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CN116064273B

CN116064273B CN202210832888.XA CN202210832888A CN116064273B CN 116064273 B CN116064273 B CN 116064273B CN 202210832888 A CN202210832888 A CN 202210832888A CN 116064273 B CN116064273 B CN 116064273B Authority

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helicobacter pylori

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standard

fma6

strain

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2022-07-14

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CN116064273A (en

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李滢

吴清平

代京莎

陈谋通

谢新强

赵辉

薛亮

王涓

丁郁

张菊梅

叶青华

吴诗

古其会

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Guangdong Kehuan Biotechnology Co ltd

Institute of Microbiology of Guangdong Academy of Sciences

Guangdong Huankai Biotechnology Co Ltd

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Guangdong Kehuan Biotechnology Co ltd

Institute of Microbiology of Guangdong Academy of Sciences

Guangdong Huankai Biotechnology Co Ltd

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2022-07-14 Application filed by Guangdong Kehuan Biotechnology Co ltd, Institute of Microbiology of Guangdong Academy of Sciences, Guangdong Huankai Biotechnology Co Ltd filed Critical Guangdong Kehuan Biotechnology Co ltd

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Abstract

本发明公开了含有特异性分子靶标的幽门螺杆菌标准菌株JMF0和FMA6及其检测和应用。HelicobacterpyloriJMF0，保藏编号为GDMCCNo：62496。HelicobacterpyloriFMA6，保藏编号为GDMCCNo：62494。本发明所述的幽门螺杆菌菌株标准物质，较目前广泛使用的标准株ATCC43504、J99有明确的生物分型信息，与标准株ATCC26695相比为不同的生物分型，可用于评价生物分型检测系统的准确性。

The invention discloses Helicobacter pylori standard strains JMFO and FMA6 containing specific molecular targets and their detection and application. Helicobacter pylori JMF0, the deposit number is GDMCCNo: 62496. Helicobacter pyloriFMA6, the deposit number is GDMCCNo: 62494. The Helicobacter pylori strain reference material of the present invention has clear biotyping information compared with the standard strains ATCC43504 and J99 widely used at present, and is a different biotyping compared with the standard strain ATCC26695, which can be used for evaluation of biotyping detection system accuracy.

Description

含有特异性分子靶标的幽门螺杆菌标准菌株JMF0和FMA6及其检测和应用Helicobacter pylori standard strains JMF0 and FMA6 containing specific molecular targets and their Detection and Application

技术领域technical field

本发明属于微生物检验技术领域，具体涉及含有特异性分子靶标的幽门螺杆菌标准菌株及其检测和应用。The invention belongs to the technical field of microbial inspection, and in particular relates to a standard strain of Helicobacter pylori containing a specific molecular target and its detection and application.

背景技术Background technique

幽门螺旋杆菌(Helicobacter pylori，HP)是一种革兰氏阴性、微需氧螺旋状细菌，与人类胃肠道疾病如胃炎、消化性溃疡、胃癌等疾病密切相关。由于越来越多的证据支持HP感染会引发胃癌患病率增加，因此在2021年，美国卫生与公众服务部(Department ofHealth and Human Services，HHS)正式将Hp认定为致癌物。据2017年《第五次全国幽门螺杆菌感染处理共识报告》，我国人群Hp感染率高达60％，其感染导致我国胃癌的发病率居高不下。因此，了解Hp的传播规律以及致病性进化是有效防控该菌所致的各类疾病的基础，而是否使用了合适的标准菌株决定了研究结果的可靠性。Helicobacter pylori (Helicobacter pylori, HP) is a Gram-negative, microaerophilic helical bacterium, which is closely related to human gastrointestinal diseases such as gastritis, peptic ulcer, gastric cancer and other diseases. As more and more evidence supports that HP infection can cause an increase in the incidence of gastric cancer, in 2021, the U.S. Department of Health and Human Services (HHS) officially recognized HP as a carcinogen. According to the "Fifth National Consensus Report on the Treatment of Helicobacter pylori Infection" in 2017, the Hp infection rate in the Chinese population is as high as 60%, and its infection leads to a high incidence of gastric cancer in my country. Therefore, understanding the transmission rules and pathogenicity evolution of Hp is the basis for effective prevention and control of various diseases caused by this bacteria, and whether the appropriate standard strains are used determines the reliability of the research results.

目前研究HP进化规律和致病性使用的标准菌株均为国外临床来源的菌株，这些菌株在基因组成、代谢特性以及致病力等多方面均与我国菌株存在差异，使我国研究者在开展HP 相关研究时缺乏可靠的参照，极大延误了我国对于HP流行规律及致病危害研究的进展。At present, the standard strains used to study the evolution law and pathogenicity of HP are all strains from foreign clinical sources. These strains are different from Chinese strains in terms of genetic composition, metabolic characteristics, and pathogenicity. The lack of reliable references for relevant research has greatly delayed the progress of research on HP prevalence and pathogenic hazards in my country.

因此，结合我国幽门螺旋杆菌高耐药、高毒力株的流行情况，开发遗传背景清晰、生理生化特性明确、基因组信息全面、耐药、毒力表型稳定、检测方法特异的标准菌株，有利于为我国临床医生及科研人员开展我国HP流行情况及防控研究，针对我国国情制定相应的HP 防控方案，提高我国国民健康水平。Therefore, in combination with the prevalence of highly drug-resistant and highly virulent strains of H. It is beneficial to conduct research on HP prevalence and prevention and control in my country for clinicians and scientific researchers in China, formulate corresponding HP prevention and control programs according to my country's national conditions, and improve the health level of our nationals.

发明内容Contents of the invention

本发明的目的在于克服现有技术的不足，提供2株幽门螺杆菌标准菌株、检测该标准菌株的特异性分子靶标和应用。本发明所提供的菌株具有稳定、典型的幽门螺杆菌生理生化特征，且能较好地反映该菌在中国地区分离株的遗传背景，具有很好的代表性。本发明的幽门螺杆菌为中国不同地区胃炎患者的分离菌株，该菌株具有典型的幽门螺杆菌生理生化特性，且能较好地反映该菌在中国地区的遗传背景。The purpose of the present invention is to overcome the deficiencies in the prior art, provide 2 strains of Helicobacter pylori standard strains, detect the specific molecular target and application of the standard strains. The bacterial strain provided by the present invention has stable and typical physiological and biochemical characteristics of Helicobacter pylori, and can better reflect the genetic background of the isolates of the bacteria in China, and is very representative. The Helicobacter pylori of the present invention is an isolated strain of gastritis patients in different regions of China. The strain has typical physiological and biochemical characteristics of Helicobacter pylori, and can better reflect the genetic background of the bacteria in China.

为了达到上述目的，本发明采取以下技术措施：In order to achieve the above object, the present invention takes the following technical measures:

2株幽门螺杆菌标准菌株的保藏信息如下：The preservation information of the two Helicobacter pylori standard strains is as follows:

幽门螺杆菌(Helicobacter pylori)JMF0于2022年5月30日保藏于广东省微生物菌种保藏中心(GDMCC)，保藏地址为：广东省广州市先烈中路100号大院59号楼5楼，邮编：510070，保藏编号为GDMCC No：62496。Helicobacter pylori (Helicobacter pylori) JMF0 was preserved in Guangdong Microbial Culture Collection Center (GDMCC) on May 30, 2022. The preservation address is: 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, postcode: 510070, the deposit number is GDMCC No: 62496.

幽门螺杆菌(Helicobacter pylori)FMA6于2022年5月27日保藏于广东省微生物菌种保藏中心(GDMCC)，保藏地址为：广东省广州市先烈中路100号大院59号楼5楼，邮编：510070，保藏编号为GDMCC No：62494。Helicobacter pylori (Helicobacter pylori) FMA6 was preserved in Guangdong Microbial Culture Collection Center (GDMCC) on May 27, 2022. The preservation address is: 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, postcode: 510070, the deposit number is GDMCC No: 62494.

本发明从中国不同地区胃炎患者胃部分离并筛选出2株幽门螺杆菌，这2株幽门螺杆菌携带特殊序列，具有特异性的分子靶标位点。The present invention isolates and screens two strains of Helicobacter pylori from the stomachs of gastritis patients in different regions of China. The two strains of Helicobacter pylori carry special sequences and have specific molecular target sites.

所述的用于检测上述幽门螺杆菌的分子靶标，包括如SEQ ID NO.3～6所示的任意一种或多种核苷酸序列。The molecular target for detecting the above-mentioned Helicobacter pylori includes any one or more nucleotide sequences as shown in SEQ ID NO.3-6.

所述如SEQ ID NO.3、SEQ ID NO.5所示的核苷酸序列的分子靶标是幽门螺杆菌JMF0 的特异性序列；所述如SEQ ID NO.4、SEQ ID NO.6所示的核苷酸序列的分子靶标是幽门螺杆菌FMA6的特异性序列。The molecular target of the nucleotide sequence shown in SEQ ID NO.3 and SEQ ID NO.5 is the specific sequence of Helicobacter pylori JMF0; the described one is shown in SEQ ID NO.4 and SEQ ID NO.6 The molecular target of the nucleotide sequence is the specific sequence of Helicobacter pylori FMA6.

本发明还提供了检测上述分子靶标的引物组，包括：针对如SEQ ID NO.5所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO.7所示的上游引物和如SEQ ID NO.8所示的下游引物；针对如SEQ ID NO.6所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO.9所示的上游引物和如SEQ ID NO.10所示的下游引物。The present invention also provides a primer set for detecting the above-mentioned molecular target, including: PCR primers for amplifying the nucleotide sequence shown in SEQ ID NO.5 include: upstream primers shown in SEQ ID NO.7 and SEQ ID NO.7 Downstream primers shown in ID NO.8; PCR primers directed at the nucleotide sequence amplification shown in SEQ ID NO.6 include: upstream primers shown in SEQ ID NO.9 and shown in SEQ ID NO.10 The downstream primers indicated.

本发明还提供了上述检测分子靶标的引物组在制备幽门螺杆菌菌株检测试剂中的用途；The present invention also provides the use of the above-mentioned primer set for detecting molecular targets in the preparation of detection reagents for Helicobacter pylori strains;

所述的分子靶标为如SEQ ID NO.3～6所示的任意一种或两种核苷酸序列；其中检测SEQ ID NO.3、5所述的分子靶标的引物用于制备检测幽门螺杆菌JMF0的检测试剂；检测SEQ ID NO.4、6所述的分子靶标的引物用于制备检测幽门螺杆菌FMA6的检测试剂。The molecular target is any one or both of the nucleotide sequences shown in SEQ ID NO.3-6; wherein the primers for detecting the molecular targets described in SEQ ID NO.3 and 5 are used to prepare and detect Helicobacter pylori A detection reagent for bacteria JMFO; primers for detecting the molecular targets described in SEQ ID NO.4 and 6 are used to prepare a detection reagent for detecting Helicobacter pylori FMA6.

优选，针对如SEQ ID NO.5所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO.7所示的上游引物和如SEQ ID NO.8所示的下游引物；针对如SEQ ID NO.6所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO.9所示的上游引物和如SEQ ID NO.10所示的下游引物。Preferably, the PCR primers for the amplification of the nucleotide sequence shown in SEQ ID NO.5 include: the upstream primer shown in SEQ ID NO.7 and the downstream primer shown in SEQ ID NO.8; The PCR primers for amplifying the nucleotide sequence shown in ID NO.6 include: the upstream primer shown in SEQ ID NO.9 and the downstream primer shown in SEQ ID NO.10.

本发明还提供了上述幽门螺杆菌JMF0和幽门螺杆菌FMA6在作为幽门螺杆菌标准菌株中的应用。The present invention also provides the application of above-mentioned Helicobacter pylori JMFO and Helicobacter pylori FMA6 as standard strains of Helicobacter pylori.

本发明与现有技术相比，本发明具有以下优点：Compared with the prior art, the present invention has the following advantages:

1、本发明所述的幽门螺杆菌菌株标准物质，较目前广泛使用的标准株ATCC43504、J99 有明确的生物分型信息，与标准株ATCC 26695相比为不同的生物分型，可用于评价生物分型检测系统的准确性。1. The Helicobacter pylori strain reference material of the present invention has clear biotyping information compared with the standard strains ATCC43504 and J99 widely used at present, and is a different biotype compared with the standard strain ATCC 26695, which can be used to evaluate biological The accuracy of the typing detection system.

2、本发明所述的幽门螺杆菌菌株标准物质，与目前广泛使用的标准株ATCC43504、ATCC 26695、J99相比，具有不同且明确的毒力基因分型，并提供了能覆盖分泌高、低毒力的两种 CagA蛋白的菌株，可用于验证Hp致癌风险的实验。2. Compared with the widely used standard strains ATCC43504, ATCC 26695 and J99, the reference material of Helicobacter pylori strains according to the present invention has different and definite virulence genotyping, and provides the ability to cover high and low secretion Two strains of CagA proteins with virulence can be used in experiments to verify the carcinogenic risk of Hp.

3、本发明所述的幽门螺杆菌菌株标准物质，与目前广泛使用的标准株ATCC43504、ATCC 26695、J99相比，提供更多抗生素的耐药表型，可作为评估克拉霉素、左氧氟沙星、甲硝唑的耐药检测系统的准确性。3. The Helicobacter pylori strain reference material of the present invention, compared with the standard strain ATCC43504, ATCC 26695, J99 widely used at present, provides the drug resistance phenotype of more antibiotics, can be used as evaluation clarithromycin, levofloxacin, formazan Accuracy of Nitazole Resistance Detection System.

4、本发明所述的幽门螺杆菌菌株标准物质，与目前广泛使用的标准株ATCC43504、ATCC 26695、J99相比，为不同的ST分型，可作为我国常见幽门螺杆菌ST分型的参考菌株。4. Compared with the widely used standard strains ATCC43504, ATCC 26695 and J99, the standard material of Helicobacter pylori strains according to the present invention has a different ST type, and can be used as a reference strain for ST typing of common Helicobacter pylori in my country .

5、本发明所述的菌株标准物质，具有特异性分子靶标，可进行快速的定性及定量检测。5. The bacterial strain reference material of the present invention has specific molecular targets and can be used for rapid qualitative and quantitative detection.

生物材料保藏biological material deposit

Helicobacter pylori JMF0于2022年5月30日保藏于广东省微生物菌种保藏中心(GDMCC)，保藏地址为：广东省广州市先烈中路100号大院59号楼5楼，邮编：510070，保藏编号为GDMCC No：62496。Helicobacter pylori JMF0 was deposited in Guangdong Microbial Culture Collection Center (GDMCC) on May 30, 2022. The preservation address is: 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, postcode: 510070, and the preservation number is GDMCC No: 62496.

Helicobacter pylori FMA6于2022年5月27日保藏于广东省微生物菌种保藏中心(GDMCC)，保藏地址为：广东省广州市先烈中路100号大院59号楼5楼，邮编：510070，保藏编号为GDMCC No：62494。Helicobacter pylori FMA6 was deposited in Guangdong Microbial Culture Collection Center (GDMCC) on May 27, 2022. The preservation address is: 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, Zip Code: 510070, and the preservation number is GDMCC No: 62494.

附图说明Description of drawings

图1是幽门螺杆菌标准株的菌落及镜检形态；Fig. 1 is the bacterium colony and microscopic examination form of Helicobacter pylori standard strain;

A1为幽门螺杆菌标准株JMF0的平板菌落形态图；A2为幽门螺杆菌标准株JMF0的镜检观察形态图；B1为幽门螺杆菌标准株FMA6的平板菌落形态图；B2为幽门螺杆菌标准株FMA6 的镜检观察形态图。A1 is the plate colony morphology of the standard Helicobacter pylori strain JMF0; A2 is the microscope observation morphology of the standard Helicobacter pylori strain JMF0; B1 is the plate colony morphology of the standard Helicobacter pylori strain FMA6; B2 is the standard Helicobacter pylori strain Microscopic observation morphology of FMA6.

图2是幽门螺杆菌标准株CagA毒素蛋白结构示意图。Fig. 2 is a schematic diagram of the structure of the CagA toxin protein of the standard Helicobacter pylori strain.

图3是幽门螺杆菌标准株与幽门螺杆菌其他标准菌株的平均核苷酸相似度分析。Figure 3 is an analysis of the average nucleotide similarity between the standard strain of Helicobacter pylori and other standard strains of Helicobacter pylori.

图4是幽门螺杆菌标准株的分子靶标验证；Figure 4 is the molecular target verification of Helicobacter pylori standard strain;

A为幽门螺杆菌标准株JMF0的分子靶标序列(SEQ ID NO.3和SEQ ID NO.5)扩增后的PCR 产物电泳图，第一泳道为菌株JMF0的电泳结果，2-23为其他幽门螺杆菌经分子靶标序列扩增后的PCR产物电泳图。结果提示除JMF0标准物质外，其余菌株均没有扩增出目的条带，表明JMF0标准物质的特有基因片段具有特异性。A is the PCR product electrophoresis figure after amplification of the molecular target sequence (SEQ ID NO.3 and SEQ ID NO.5) of the Helicobacter pylori standard strain JMF0, the first swimming lane is the electrophoresis result of the bacterial strain JMF0, and 2-23 are other pylori Electrophoresis of PCR products of Helicobacter amplified by molecular target sequence. The results indicated that except for the JMF0 standard substance, the other strains did not amplify the target band, indicating that the unique gene fragment of the JMF0 standard substance was specific.

B为幽门螺杆菌标准株FMA6的分子靶标序列(SEQ ID NO.4和SEQ ID NO.6)扩增后的PCR 产物电泳图，第一泳道为菌株JMF0的电泳结果，2-23为其他幽门螺杆菌经分子靶标序列扩增后的PCR产物电泳图。结果提示除FMA6标准物质外，其余菌株均没有扩增出目的条带，表明FMA6标准物质的特有基因片段具有特异性。B is the PCR product electrophoresis after the amplification of the molecular target sequence (SEQ ID NO.4 and SEQ ID NO.6) of the Helicobacter pylori standard strain FMA6, the first swimming lane is the electrophoresis result of the bacterial strain JMF0, and 2-23 are other pylori Electrophoresis of PCR products of Helicobacter amplified by molecular target sequence. The results indicated that except for the FMA6 standard substance, the other strains did not amplify the target band, indicating that the specific gene fragment of the FMA6 standard substance was specific.

图5中A为应用特征引物序列检测不同浓度幽门螺杆菌标准株JMF0的qPCR扩增情况；B 为应用特征引物检测幽门螺杆菌标准株JMF0定量的标准曲线；C为应用特征引物序列检测不同浓度幽门螺杆菌标准株FMA6的qPCR扩增情况；D为应用特征引物检测幽门螺杆菌标准株FMA6定量的标准曲线。In Fig. 5, A is the qPCR amplification of different concentrations of Helicobacter pylori standard strain JMF0 using characteristic primer sequences; B is the standard curve of using characteristic primer sequences to detect the quantification of Helicobacter pylori standard strain JMF0; C is using characteristic primer sequences to detect different concentrations The qPCR amplification of the Helicobacter pylori standard strain FMA6; D is the standard curve for the quantitative detection of the Helicobacter pylori standard strain FMA6 using characteristic primers.

具体实施方式Detailed ways

为更清楚地表述本发明的技术方案，下面结合具体实施例进一步说明，但不能用于限制本发明，此仅是本发明的部分实施例。In order to describe the technical solution of the present invention more clearly, it will be further described below in conjunction with specific examples, but it cannot be used to limit the present invention, which are only some examples of the present invention.

下述实施例中涉及的培养基及试剂如下：The medium and reagents involved in the following examples are as follows:

抗生素混悬液的制备：万古霉素10mg/mL、两性霉素B 5mg/mL、甲氧苄氨嘧啶20mg/mL、头孢磺啶10mg/mL，将上述抗生素按其含量溶于灭菌水中，混合均匀。Preparation of antibiotic suspension: vancomycin 10mg/mL, amphotericin B 5mg/mL, trimethoprim 20mg/mL, cefsulodin 10mg/mL, dissolve the above antibiotics in sterilized water according to their content, well mixed.

幽门螺杆菌分离培养基(g/L)：酵母浸粉3.0g/L、酪胨12.0g/L、动物组织消化物5.0g/L、牛肉浸粉3.0g/L、玉米淀粉1.0g/L、氯化钠5.0g/L、琼脂13.5g/L、无菌绵羊红细胞或小牛血清100mL/L、抗生素混悬液10mL/L，将上述成分按其含量溶于灭菌水中，混合均匀。Helicobacter pylori isolation medium (g/L): Yeast extract powder 3.0g/L, casetone 12.0g/L, animal tissue digestate 5.0g/L, beef extract powder 3.0g/L, corn starch 1.0g/L , sodium chloride 5.0g/L, agar 13.5g/L, sterile sheep red blood cells or calf serum 100mL/L, antibiotic suspension 10mL/L, dissolve the above ingredients in sterilized water according to their content, and mix well.

幽门螺杆菌增殖培养基(g/L)：酵母浸粉2.02g/L、胰蛋白胨10.52g/L、葡萄糖1.56g/L、亚硫酸氢钠0.10g/L、可溶性淀粉10.0g/L、氯化钠5.0g/L、小牛血清100mL/L、抗生素混悬液10mL/L，将上述成分按其含量溶于灭菌水中，混合均匀。Helicobacter pylori proliferation medium (g/L): yeast extract powder 2.02g/L, tryptone 10.52g/L, glucose 1.56g/L, sodium bisulfite 0.10g/L, soluble starch 10.0g/L, chlorine Sodium chloride 5.0g/L, calf serum 100mL/L, antibiotic suspension 10mL/L, dissolve the above ingredients in sterilized water according to their content, and mix well.

幽门螺杆菌保存液(g/L)：蛋白胨4.0g/L、酵母浸膏0.8g/L、胰酪蛋白胨4.0g/L、氯化钠2.0g/L、葡萄糖0.4g/L、亚硫酸氢钠0.04g/L、偏重亚硫酸钠0.1g/L、七水合硫酸亚铁0.1g/L、丙酮酸钠0.1g/L、丙三醇200mL/L，将上述成分按其含量溶于灭菌水中，混合均匀。Helicobacter pylori preservation solution (g/L): peptone 4.0g/L, yeast extract 0.8g/L, tryptone 4.0g/L, sodium chloride 2.0g/L, glucose 0.4g/L, hydrogen sulfite Sodium 0.04g/L, sodium metabisulfite 0.1g/L, ferrous sulfate heptahydrate 0.1g/L, sodium pyruvate 0.1g/L, glycerol 200mL/L, dissolve the above ingredients in sterilized water according to their content, well mixed.

实施例1幽门螺杆菌标准株的分离与培养Isolation and cultivation of embodiment 1 Helicobacter pylori standard strain

1.1幽门螺杆菌的分离与培养1.1 Isolation and cultivation of Helicobacter pylori

(1)幽门螺杆菌的分离与保藏：胃镜下采集我国各省市罹患胃部疾病患者的胃粘膜组织。在无菌环境下，取新鲜胃粘膜样品涂布于幽门螺杆菌分离培养基上，后放置于37℃微需氧环境中(5％O₂、10％CO₂、85％N₂)培养5～7天。挑取平板上形态典型的菌落至幽门螺杆菌分离培养基平板上进行划线纯化，纯化2次后挑取单菌落接种入幽门螺杆菌增殖培养基中，37℃ 微需氧条件培养2天。将400μL菌液加入600μL幽门螺杆菌保存液中轻轻吹打混匀，保藏于 -80℃超低温冰箱中。其中幽门螺杆菌标准菌株JMF0分离自广东地区一位无胃癌家族史的慢性胃炎患者，标准菌株FMA6分离自福建地区一位有胃癌家族史的胃炎患者。(1) Isolation and preservation of Helicobacter pylori: Gastric mucosal tissues from patients with gastric diseases in various provinces and cities in my country were collected under gastroscope. In a sterile environment, fresh gastric mucosa samples were spread on Helicobacter pylori isolation medium, and placed in a microaerobic environment (5% O ₂ , 10% CO ₂ , 85% N ₂ ) at 37°C for 5 ~7 days. Pick the colonies with typical morphology on the plate and put them on the Helicobacter pylori isolation medium plate for streak purification. After purification twice, pick a single colony and inoculate it into the Helicobacter pylori proliferation medium, and culture it under microaerophilic conditions at 37°C for 2 days. Add 400 μL of bacterial solution to 600 μL of Helicobacter pylori preservation solution and gently blow and mix, and store in a -80°C ultra-low temperature refrigerator. The Helicobacter pylori standard strain JMF0 was isolated from a chronic gastritis patient without a family history of gastric cancer in Guangdong, and the standard strain FMA6 was isolated from a gastritis patient with a family history of gastric cancer in Fujian.

(2)幽门螺杆菌的复苏与培养：从保藏管中将蘸取10μL幽门螺杆菌标准株保藏液，涂布于幽门螺杆菌分离培养基上，在37℃微需氧环境中培养2～4天后，获得复苏的幽门螺杆菌标准株，其在平板上呈针尖大小的半透明菌落。从含复苏的幽门螺杆菌的平板上挑取单菌落，接种于幽门螺杆菌增殖培养基中，37℃微需氧环境中培养2～4天，获得增殖的幽门螺杆菌标准株菌液。(2) Recovery and cultivation of Helicobacter pylori: dip 10 μL of Helicobacter pylori standard strain preservation solution from the storage tube, spread it on the Helicobacter pylori isolation medium, and culture it in a microaerobic environment at 37°C for 2-4 days. Two days later, the revived Helicobacter pylori standard strain was obtained, which appeared as pinpoint-sized translucent colonies on the plate. Pick a single colony from the plate containing the revived Helicobacter pylori, inoculate it in the Helicobacter pylori proliferation medium, and culture it in a microaerobic environment at 37°C for 2 to 4 days to obtain the proliferated Helicobacter pylori standard strain bacterial liquid.

(3)幽门螺杆菌的分子鉴定：采用细菌DNA提取试剂盒(环凯生物，中国)提取幽门螺旋杆菌DNA，后采用2×PCRmix(环凯生物，中国)进行聚合酶链式反应(Polymerase ChainReaction，PCR)扩增。PCR扩增引物采用16SrRNA基因通用引物，上游引物序列为27F：5’ -AGA GTT TGA TCC TGG CTC AG-3’；下游引物序列为1492R：5’-CTA CGG CTA CCT TGT TACGA-3’。PCR反应条件为：预变性95℃5min；95℃30s，56℃30s，72℃1min30s共35 个循环，72℃退火延伸10min。对PCR产物进行切胶回收，后进行一代测序(由天一辉远科技有限公司完成)。测序后获得幽门螺杆菌JMF0的16S rRNA基因序列如SEQ ID No.11所示，获得幽门螺杆菌FMA6的16S rRNA基因序列如SEQ ID No.12所示。获得的16S rRNA基因序列上传NCBI数据库(https://blast.ncbi.nlm.nih.gov)，结果提示幽门螺杆菌标准菌株JMF0与幽门螺杆菌ATCC 43504同源性最高，16S rRNA相似度为99.40％，,命名为幽门螺旋杆菌(Helicobacter pylori)JMF0；幽门螺杆菌标准菌株FMA6与幽门螺杆菌ATCC 43504同源性最高，16S rRNA相似度为99.33％，，命名为幽门螺旋杆菌(Helicobacter pylori)FMA6。(3) Molecular identification of Helicobacter pylori: Helicobacter pylori DNA was extracted using a bacterial DNA extraction kit (Huankai Biotechnology, China), and then polymerase chain reaction (Polymerase Chain Reaction) was carried out using 2×PCRmix (Huankai Biotechnology, China). , PCR) amplification. 16S rRNA gene general primers were used as primers for PCR amplification. The upstream primer sequence was 27F: 5'-AGA GTT TGA TCC TGG CTC AG-3'; the downstream primer sequence was 1492R: 5'-CTA CGG CTA CCT TGT TACGA-3'. The PCR reaction conditions were: pre-denaturation at 95°C for 5min; 35 cycles of 95°C for 30s, 56°C for 30s, 72°C for 1min and 30s, and annealing at 72°C for 10min. The PCR products were gel-cut and recovered, followed by next-generation sequencing (completed by Tianyi Huiyuan Technology Co., Ltd.). After sequencing, the 16S rRNA gene sequence of Helicobacter pylori JMF0 is shown as SEQ ID No.11, and the 16S rRNA gene sequence of Helicobacter pylori FMA6 is shown as SEQ ID No.12. The obtained 16S rRNA gene sequence was uploaded to the NCBI database (https://blast.ncbi.nlm.nih.gov), and the results indicated that the standard Helicobacter pylori strain JMF0 had the highest homology with Helicobacter pylori ATCC 43504, and the 16S rRNA similarity was 99.40 %, named Helicobacter pylori (Helicobacter pylori) JMF0; Helicobacter pylori standard strain FMA6 has the highest homology with Helicobacter pylori ATCC 43504, 16S rRNA similarity is 99.33%, named Helicobacter pylori (Helicobacter pylori) FMA6 .

实施例2幽门螺杆菌标准株的生理、生化特征鉴定Physiological and biochemical characteristic identification of embodiment 2 Helicobacter pylori standard strain

(1)幽门螺杆菌标准株在培养基上的形态特征：在幽门螺杆菌增殖培养平板上，标准菌株的菌落呈透明针尖样，当接种密度较高时，可在平板上形成半透明菌苔，如图1所示。(1) Morphological characteristics of the standard strain of Helicobacter pylori on the culture medium: on the Helicobacter pylori proliferation culture plate, the colony of the standard strain is transparent needle-like, and when the inoculation density is high, a translucent bacterial lawn can be formed on the plate ,As shown in Figure 1.

(2)幽门螺杆菌标准株在光镜下的形态特征：采用革兰氏染色对幽门螺杆菌标准株的光镜下形态进行观察。革兰氏染色后幽门螺杆菌标准菌株呈红色，形态为S状弯曲状、短棒状、球型或者丝型，菌株在空气中暴露时间越长，标准菌株的球型体检出比例越高，如图1所示。(2) Morphological characteristics of the standard strain of Helicobacter pylori under the light microscope: Gram staining was used to observe the morphology of the standard strain of Helicobacter pylori under the light microscope. After Gram staining, the standard strain of Helicobacter pylori is red, and the shape is S-shaped curved, short rod, spherical or filamentous. The longer the strain is exposed to the air, the higher the detection ratio of the standard strain is. Figure 1 shows.

(3)幽门螺杆菌标准株的生化代谢特征：(3) Biochemical and metabolic characteristics of Helicobacter pylori standard strain:

采用梅里埃弯曲菌鉴定试剂盒API Campy或类似的检测试剂盒对幽门螺杆菌标准株的生化代谢特征进行检测，结果如表1所示。比对API生化鉴定系统，提示菌株为幽门螺杆菌；但菌株典型值T指数仅0.5，其中HIP与来自澳洲的ATCC 43504标准株有较大差异，是具有中国地区遗传背景和典型性状的菌株，因此这些菌株可以作为中国幽门螺杆菌检测的特异性参考菌株。The biochemical and metabolic characteristics of the standard strain of Helicobacter pylori were detected by the Campylobacter Mérieux identification kit API Campy or similar detection kits, and the results are shown in Table 1. Compared with the API biochemical identification system, it was suggested that the strain was Helicobacter pylori; however, the typical T index of the strain was only 0.5, and HIP was quite different from the ATCC 43504 standard strain from Australia. It was a strain with a genetic background and typical traits in China. Therefore, these strains can be used as specific reference strains for the detection of Helicobacter pylori in China.

表1幽门螺杆菌标准株的生化代谢特性Table 1 Biochemical metabolic characteristics of Helicobacter pylori standard strain

(4)幽门螺杆菌标准株的酶学特征：(4) Enzymatic characteristics of Helicobacter pylori standard strain:

采用梅里埃API ZYM酶学检测或类似的检测试剂盒对幽门螺杆菌标准株的酶学特征进行检测，结果如表2所示。The enzymatic characteristics of Helicobacter pylori standard strains were detected by Mérieux API ZYM enzyme detection kit or similar detection kit, and the results are shown in Table 2.

表2幽门螺杆菌标准株的酶学特性Table 2 Enzymatic characteristics of Helicobacter pylori standard strain

根据梅里埃API ZYM酶学检测结果对幽门螺杆菌标准株进行生物学分型，结果提示 JMF0为IIa型生物分型，而FMA6为不典型的生物分型。据文献，幽门螺杆菌ATCC 26695的生物学分型为I型，提示JMF0及FMA6可以作为其它生物型检测的特异性参考菌株。According to the results of Mérieux API ZYM enzymatic detection, the standard strain of Helicobacter pylori was biologically typed, and the results indicated that JMF0 was a type IIa biotype, while FMA6 was an atypical biotype. According to literature, the biological type of Helicobacter pylori ATCC 26695 is type I, suggesting that JMF0 and FMA6 can be used as specific reference strains for detection of other biological types.

实施例3幽门螺杆菌标准株的毒力特征The virulence characteristic of embodiment 3 Helicobacter pylori standard strain

(1)幽门螺杆菌标准株的毒力因子携带情况(1) Carrying of virulence factors of Helicobacter pylori standard strain

参照说明书方法采用细菌基因组提取试剂盒提取幽门螺杆菌标准株的基因组DNA。采用 PCR方法对幽门螺杆菌标准株的毒力因子携带情况进行检测。检测所使用的引物由广州天一辉远基因科技有限公司合成，引物序列见表3。采用Takara的MaxDNA Polymerase进行PCR反应，反应体系配置方法为：PrimeSTAR Max Premix(2×)12.5μL，引物各1μL，模板1μL，超纯水9.5μL；PCR的反应条件为98℃预变性5min，后以98℃变性10s、55℃退火5s、72℃延伸5s共35个循环，最后72℃终延伸10min。PCR结束后进行琼脂糖凝胶电泳，确认是否存在相应的毒力基因扩增产物及产物条带大小是否正确。Genomic DNA of the standard strain of Helicobacter pylori was extracted using the bacterial genome extraction kit according to the method in the manual. The PCR method was used to detect the carrying status of the virulence factors of the standard strain of Helicobacter pylori. The primers used in the detection were synthesized by Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd., and the primer sequences are shown in Table 3. Using Takara MaxDNA Polymerase for PCR reaction, the reaction system configuration method is: PrimeSTAR Max Premix (2×) 12.5 μL, each primer 1 μL, template 1 μL, ultrapure water 9.5 μL; Denaturation for 10 s, annealing at 55°C for 5 s, extension at 72°C for 5 s, a total of 35 cycles, and a final extension at 72°C for 10 min. After PCR, agarose gel electrophoresis was performed to confirm whether there were corresponding virulence gene amplification products and whether the product band size was correct.

经PCR及琼脂糖凝胶电泳确认，所述HP标准菌株JMF0携带的毒力因子有：ureA、vacA、 cagA、oipA、babA、napA。Confirmed by PCR and agarose gel electrophoresis, the virulence factors carried by the HP standard strain JMF0 include: ureA, vacA, cagA, oipA, babA, napA.

HP标准菌株FMA6携带的毒力因子有：ureA、vacA、cagA、oipA、babA、napA。The virulence factors carried by HP standard strain FMA6 are: ureA, vacA, cagA, oipA, babA, napA.

表3幽门螺杆菌毒力基因检测引物及目标基因检测条带Table 3 Helicobacter pylori virulence gene detection primers and target gene detection bands

(2)幽门螺杆菌标准株cagA蛋白的分型情况(2) Typing of the cagA protein of the Helicobacter pylori standard strain

据多项研究指出，细胞毒素相关蛋白A(cytotoxin-associated gene A,cagA)蛋白是幽门螺杆菌致癌的重要毒力因子，不同的幽门螺杆菌根据其是否分泌cagA蛋白、分泌不同构型的 cagA蛋白分为不同的致癌风险。据文献，幽门螺杆菌ATCC 26695、ATCC 43504均分泌西方型的cagA毒素，而J99则不分泌cagA毒素。采用高通量测序对幽门螺杆菌标准株进行全基因组检测，检测后注释相关毒力蛋白。分析CagA蛋白的氨基酸序列，如SEQ IDNO.1～2所示。分析如图2所示，幽门螺杆菌标准株JMF0的cagA为西方型，而FMA6为东亚型。结果提示JMF0及FMA6具有更系统的毒力背景信息，更全面地覆盖幽门螺杆菌致病及致癌机制研究的需求。According to a number of studies, cytotoxin-associated gene A (cagA) protein is an important virulence factor for Helicobacter pylori carcinogenesis. Different Helicobacter pylori secrete cagA protein and secrete different configurations of cagA Proteins are grouped into different cancer risk groups. According to the literature, Helicobacter pylori ATCC 26695 and ATCC 43504 both secrete Western-type cagA toxin, while J99 does not secrete cagA toxin. High-throughput sequencing was used to detect the whole genome of the standard strain of Helicobacter pylori, and the relevant virulence proteins were annotated after detection. The amino acid sequence of the CagA protein was analyzed, as shown in SEQ ID NO.1-2. As shown in Figure 2, the cagA of the standard Helicobacter pylori strain JMF0 is the Western type, while FMA6 is the East Asian type. The results suggest that JMF0 and FMA6 have more systematic virulence background information and more comprehensively cover the needs of research on the pathogenic and carcinogenic mechanisms of Helicobacter pylori.

实施例4幽门螺杆菌标准株的基因组特征Genome characteristics of embodiment 4 Helicobacter pylori standard strain

(1)幽门螺杆菌标准株的全基因组测序(1) Whole genome sequencing of standard Helicobacter pylori strain

参照说明书方法采用细菌基因组DNA提取试剂盒对幽门螺杆菌标准株进行基因组DNA 提取。采用Illumina Nextseq 550二代测序及Nanopore MinION三代测序平台对幽门螺杆菌标准株进行全基因组测序。二代测序采用AMT Rapid DNA-Seq Kit for Illumina(CISTRO,CHINA) 建库，采用High Output v2.5 kit(Illumina,USA)试剂盒测序。三代测序采用Rapid Barcoding Sequencing Kit(Nanopore,UK)建库，后采用R9.4.1芯片(Nanopore,UK)测序。下机数据分别采用Trimmomatic(v0.39)及Filtlong(v0.2.0)软件进行质控，后采用Unicycler(v0.4.8)软件进行组装。组装后幽门螺杆菌的基因组采用Quast(v5.0.2)软件进行基因组质控评估，采用Prokka(v1.13)软件进行注释，采用Pyani(v0.2.9)进行平均核苷酸相似度(Average Nucleotide Identity，ANI)分析。Genomic DNA was extracted from the Helicobacter pylori standard strain using the bacterial genomic DNA extraction kit according to the method in the manual. The whole genome of the standard Helicobacter pylori strain was sequenced using the Illumina Nextseq 550 next-generation sequencing and the Nanopore MinION third-generation sequencing platform. AMT Rapid DNA-Seq Kit for Illumina (CISTRO, CHINA) was used for next-generation sequencing, and High Output v2.5 kit (Illumina, USA) was used for sequencing. The third-generation sequencing used the Rapid Barcoding Sequencing Kit (Nanopore, UK) to construct the library, and then used the R9.4.1 chip (Nanopore, UK) for sequencing. Trimmomatic (v0.39) and Filtlong (v0.2.0) software were used for quality control of the off-machine data, and Unicycler (v0.4.8) software was used for assembly. After assembly, the genome of Helicobacter pylori was evaluated by Quast (v5.0.2) software for genome quality control, Prokka (v1.13) software was used for annotation, and Pyani (v0.2.9) was used for average nucleotide similarity (Average Nucleotide Identity , ANI) analysis.

(2)幽门螺杆菌标准株的全基因组特征(2) Genome-wide characteristics of Helicobacter pylori standard strain

基因组分析提示标准菌株JMF0的基因组大小为1.575Mb，GC含量为38.88％；JMF0基因组中有1568个编码蛋白，35个tRNA，6个核糖体RNA；在JMF0的1568个编码蛋白中，其中有1199个蛋白为功能已知的蛋白，369个为假想蛋白；在已知功能的基因中，316个与微生物代谢相关，174个与蛋白质翻译相关，118个与能量代谢相关，54个与应激反应、防御及毒力相关，47个与RNA转录相关，82个与细胞代谢相关，54个与DNA转录、复制相关，36个与细胞膜转运相关，19个与细胞被膜相关，2个与细胞调节和信号传导相关。如附图3A所示，ANI分析显示JMF0与已报道的各类分型幽门螺杆菌基因组平均核苷酸相似度均较低，JMF0与数据库中已报到的东亚分型、来自日本的F16菌株相似度最高，但相似度亦仅为96.32％，与非洲分型、来自赞比亚的Gambia94菌株相似度最低，为92.75％。结果提示是JMF0与各地区已报到的标准菌株在基因组上有较大差异，因此相比于来自澳洲的ATCC26695、ATCC 43504及来自非洲的J99更适合作为作为中国幽门螺杆菌检测的特异性参考菌株。Genome analysis indicated that the genome size of the standard strain JMF0 was 1.575Mb, and the GC content was 38.88%; there were 1568 encoded proteins, 35 tRNAs, and 6 ribosomal RNAs in the JMF0 genome; among the 1568 encoded proteins of JMF0, 1199 4 proteins are proteins with known functions, and 369 are hypothetical proteins; among the genes with known functions, 316 are related to microbial metabolism, 174 are related to protein translation, 118 are related to energy metabolism, and 54 are related to stress response , defense and virulence, 47 are related to RNA transcription, 82 are related to cell metabolism, 54 are related to DNA transcription and replication, 36 are related to cell membrane transport, 19 are related to cell envelope, 2 are related to cell regulation and related to signal transduction. As shown in Figure 3A, ANI analysis shows that the average nucleotide similarity between JMF0 and the reported various types of Helicobacter pylori genomes is low, and JMF0 is similar to the reported East Asian type and F16 strain from Japan in the database The degree of similarity is the highest, but the similarity is only 96.32%, and the similarity with African strain Gambia94 from Zambia is the lowest, which is 92.75%. The results suggest that JMF0 has a large genome difference with the standard strains reported in various regions, so it is more suitable as a specific reference strain for the detection of Helicobacter pylori in China than ATCC26695, ATCC 43504 from Australia and J99 from Africa .

基因组分析提示标准菌株FMA6的基因组大小为1.563Mb，GC含量为38.88％；FMA6基因组中有1579个编码蛋白，34个tRNA，6个核糖体RNA；在FMA6的1579个编码蛋白中，其中有1183个蛋白为功能已知的蛋白，396个为假想蛋白；在已知功能的基因中，321 个与微生物代谢相关，175个与蛋白质翻译相关，118个与能量代谢相关，54个与应激反应、防御及毒力相关，47个与RNA转录相关，82个与细胞代谢相关，53个与DNA转录、复制相关，34个与细胞膜转运相关，19个与细胞被膜相关，2个与细胞调节和信号传导相关。如图3B所示，ANI分析显示FMA6与已报道的各类分型幽门螺杆菌基因组平均核苷酸相似度均较低，FMA6与数据库中已报到的东亚分型、来自日本的35A菌株相似度最高，但相似度亦仅为96.71％，与非洲分型、来自赞比亚的Gambia94菌株相似度最低，为92.67％。结果提示是FMA6与各地区已报到的标准菌株在基因组上有较大差异，因此相比于来自澳洲的ATCC 26695、ATCC43504及来自非洲的J99更适合作为中国幽门螺杆菌检测的特异性参考菌株。Genome analysis indicated that the genome size of the standard strain FMA6 was 1.563Mb, and the GC content was 38.88%; there were 1579 encoded proteins, 34 tRNAs, and 6 ribosomal RNAs in the FMA6 genome; among the 1579 encoded proteins of FMA6, 1183 4 proteins are proteins with known functions, and 396 are hypothetical proteins; among the genes with known functions, 321 are related to microbial metabolism, 175 are related to protein translation, 118 are related to energy metabolism, and 54 are related to stress response , defense and virulence, 47 are related to RNA transcription, 82 are related to cell metabolism, 53 are related to DNA transcription and replication, 34 are related to cell membrane transport, 19 are related to cell envelope, 2 are related to cell regulation and related to signal transduction. As shown in Figure 3B, ANI analysis shows that the average nucleotide similarity between FMA6 and the reported types of Helicobacter pylori genomes is low, and the similarity between FMA6 and the East Asian type and 35A strain from Japan reported in the database The highest, but the similarity is only 96.71%, and the lowest similarity with the African type, Gambia94 strain from Zambia, is 92.67%. The results suggest that FMA6 has a large genome difference with the standard strains reported in various regions, so it is more suitable as a specific reference strain for the detection of Helicobacter pylori in China than ATCC 26695, ATCC 43504 from Australia and J99 from Africa.

实施例5幽门螺杆菌标准株的抗生素药敏特征Antibiotic susceptibility characteristics of embodiment 5 Helicobacter pylori standard strain

采用琼脂稀释法对幽门螺杆菌标准株进行药敏检测，采用幽门螺旋杆菌ATCC43504作为质控菌株。检测根据欧洲抗菌药物敏感性试验委员会(The EuropeanCommittee on Antimicrobial Susceptibility Testing，EUCAST)制定的标准进行结果判定。将阿莫西林、克拉霉素、左氧氟沙星、甲硝唑及四环素五种抗生素配置成抗生素贮存液，根据所需检测的药敏浓度，分别加至幽门螺杆菌增殖培养基中，制成含相应浓度抗生素的幽门螺杆菌药敏检测平板，以检测抗生素对幽门螺杆菌标准株的最小抑菌浓度(Minimum Inhibitory Concentration， MIC)。将待测幽门螺杆菌标准株菌悬液浓度调整为1×10⁷cfu/mL，接种2μL菌悬液至含相应浓度抗生素的检测平板上，于37℃微需氧环境中培养72小时，观察含相应浓度抗生素的检测平板上有无幽门螺杆菌菌落形成。以不能形成菌落的平板中的最低抗生素浓度为该种抗生素对幽门螺杆菌标准株的MIC值。每个药物浓度进行平行操作3次，得出MIC的平均值。两株幽门螺杆菌标准菌株的MIC值如表4所示，根据EUCAST标准，标准菌株JMF0的耐药谱为：左氧氟沙星、甲硝唑；标准菌株FMA6的耐药谱为：甲硝唑。相比于来自澳洲的ATCC 26695(对所有抗生素敏感)、ATCC 43504(耐受克拉霉素)及来自非洲的J99(无耐受抗生素信息)，幽门螺杆菌JMF0及FMA6可以作为评价耐药检测系统的准确性，亦可以作为研发抗耐药性幽门螺杆菌的底物。The standard strain of Helicobacter pylori was tested for drug susceptibility by agar dilution method, and Helicobacter pylori ATCC43504 was used as the quality control strain. The test results were judged according to the standards established by The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Five antibiotics, amoxicillin, clarithromycin, levofloxacin, metronidazole and tetracycline, were formulated as antibiotic stock solutions, and were added to the Helicobacter pylori proliferation medium according to the drug sensitivity concentration to be detected to make Antibiotic Helicobacter pylori susceptibility testing plate to detect the minimum inhibitory concentration (Minimum Inhibitory Concentration, MIC) of antibiotics to the Helicobacter pylori standard strain. Adjust the concentration of the standard strain of Helicobacter pylori to be tested to 1×10 ⁷ cfu/mL, inoculate 2 μL of the bacterial suspension onto the detection plate containing the corresponding concentration of antibiotics, and culture it in a microaerophilic environment at 37°C for 72 hours. Whether there is Helicobacter pylori colony formation on the detection plate containing the corresponding concentration of antibiotics. The MIC value of the antibiotic against the Helicobacter pylori standard strain was defined as the lowest antibiotic concentration in the plate that could not form colonies. Each drug concentration was performed in parallel three times to obtain the average value of MIC. The MIC values of the two standard strains of Helicobacter pylori are shown in Table 4. According to the EUCAST standard, the drug resistance spectrum of the standard strain JMF0 is: levofloxacin and metronidazole; the drug resistance spectrum of the standard strain FMA6 is: metronidazole. Compared with ATCC 26695 from Australia (susceptible to all antibiotics), ATCC 43504 (resistant to clarithromycin) and J99 from Africa (no resistance antibiotic information), Helicobacter pylori JMF0 and FMA6 can be used as a detection system for evaluating drug resistance It can also be used as a substrate for the development of drug-resistant Helicobacter pylori.

表4幽门螺杆菌标准株对不同抗生素的MIC值Table 4 Helicobacter pylori standard strains to the MIC value of different antibiotics

实施例6幽门螺杆菌标准株的多位点序列分型特征The multi-locus sequence typing characteristic of embodiment 6 Helicobacter pylori standard strain

多位点序列分型(multilocus sequence typing，MLST)是一种基于核酸序列测定的细菌分型方法。采用MLST官网提供的实验方案，对幽门螺杆菌标准株进行7个管家基因的Sanger 测序。引物序列见表5，PCR反应体系配置及反应条件同实施例3。PCR结束后进行凝胶电泳，确认产物条带大小正确，切胶后PCR产物进行Sanger测序。测序结果与MLST数据库中进行匹配分析，分别获取7个管家基因的等位基因数值，并形成相应的等位基因谱，判断其序列型。两株幽门螺杆菌标准株的ST型如表6所示，2株幽门螺杆菌标准株均为新的ST型别，与来自澳洲的ATCC 26695、ATCC 43504及来自非洲的J99的ST型不同，是具有中国地区遗传背景和典型性状的菌株，因此这些菌株可以作为中国幽门螺杆菌ST4039和 ST4040检测的特异性参考菌株。Multilocus sequence typing (MLST) is a bacterial typing method based on nucleic acid sequence determination. Using the experimental protocol provided by the MLST official website, Sanger sequencing of seven housekeeping genes was performed on the standard strain of Helicobacter pylori. The primer sequences are shown in Table 5, and the PCR reaction system configuration and reaction conditions are the same as in Example 3. After PCR, gel electrophoresis was performed to confirm that the product band size was correct, and the PCR product was subjected to Sanger sequencing after gel cutting. The sequencing results were matched with the MLST database to obtain the allelic values of the seven housekeeping genes, and form the corresponding allelic spectrum to determine the sequence type. The ST types of the two Helicobacter pylori standard strains are shown in Table 6. The two Helicobacter pylori standard strains are all new ST types, which are different from the ST types of ATCC 26695, ATCC 43504 from Australia and J99 from Africa. They are strains with genetic background and typical traits in China, so these strains can be used as specific reference strains for the detection of Helicobacter pylori ST4039 and ST4040 in China.

表5幽门螺杆菌MLST分型引物序列Table 5 Helicobacter pylori MLST typing primer sequence

表6幽门螺杆菌标准株MLST分型结果Table 6 Helicobacter pylori standard strain MLST typing results

实施例7幽门螺杆菌标准株的特性序列分析The characteristic sequence analysis of embodiment 7 Helicobacter pylori standard strain

根据幽门螺杆菌的泛基因组分析结果获得菌株特有的非必需基因。共选取1630株幽门螺杆菌的基因组序列来进行泛基因组分析。泛基因组采用原核生物泛基因组自动化分析软件 (Pan-Genomics Analysis Pipeline，PGAP)中的GF方法来分析，通过本地Perl脚本对分析结果进行处理，得到所有菌株的核心基因及非核心基因信息。Strain-specific non-essential genes were obtained from the results of pan-genome analysis of Helicobacter pylori. A total of 1630 genome sequences of Helicobacter pylori were selected for pan-genome analysis. The pan-genome was analyzed using the GF method in the prokaryotic pan-genome automated analysis software (Pan-Genomics Analysis Pipeline, PGAP), and the analysis results were processed through local Perl scripts to obtain core and non-core gene information of all strains.

提取幽门螺杆菌特有的非核心基因蛋白序列，通过本地Blast分别将其比对至幽门螺杆菌的蛋白总库及NCBI非冗余蛋白数据库(Non-Redundant Protein SequenceDatabase，NR)，去除能比对到NR库的序列，剩余序列即为特异性序列。JMF0的特异基因如SEQ ID NO.3所示，FMA6的特异基因如SEQ ID NO.4所示。使用Primer BLAST(https://www.ncbi.nlm.nih.g ov/tools/primer-blast/)针对JMF0的特异基因如SEQ ID NO.3基因设计引物，获得正向引物SE Q ID NO.7及反向引物SEQ ID NO.8；针对FMA6的特异基因如SEQ ID NO.4基因设计引物，获得正向引物SEQ ID NO.9及反向引物SEQ ID NO.10。由广州天一辉远基因科技有限公司合成相应引物，按实施例3配置PCR反应体系，PCR的反应条件为98℃预变性5min，后以98℃ 变性10s、60℃退火5s、72℃延伸5s共35个循环，最后72℃终延伸10min；其中JMF0及FMA6 的退火温度为68℃，GZCA3的退火温度为55℃。特有基因通过PCR扩增后，可采用琼脂糖凝胶电泳检验其特异性，该分子序列在其他幽门螺杆菌中的特异性具体PCR结果见图4。同时， PCR产物经Sanger测序可获得特异性序列，幽门螺杆菌JMF0的特异序列如SEQ.ID No.5所示，幽门螺杆菌FMA6的特异序列如SEQ.ID No.6所示，这些特异性序列同样具有识别标准菌株的作用。Extract the non-core gene protein sequence unique to Helicobacter pylori, and compare it to the total protein library of Helicobacter pylori and the NCBI non-redundant protein database (Non-Redundant Protein Sequence Database, NR) through local Blast, and remove those that cannot be compared to The sequence of the NR library, the remaining sequence is the specific sequence. The specific gene of JMF0 is shown in SEQ ID NO.3, and the specific gene of FMA6 is shown in SEQ ID NO.4. Use Primer BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) to design primers for the specific gene of JMF0 such as SEQ ID NO.3 gene to obtain forward primer SEQ ID NO. 7 and reverse primer SEQ ID NO.8; primers were designed for a specific gene of FMA6 such as the gene of SEQ ID NO.4 to obtain forward primer SEQ ID NO.9 and reverse primer SEQ ID NO.10. The corresponding primers were synthesized by Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd., and the PCR reaction system was configured according to Example 3. The PCR reaction conditions were pre-denaturation at 98°C for 5 minutes, followed by denaturation at 98°C for 10 seconds, annealing at 60°C for 5 seconds, and extension at 72°C for 5 seconds. A total of 35 cycles, with a final extension of 10 min at 72°C; the annealing temperature of JMFO and FMA6 was 68°C, and the annealing temperature of GZCA3 was 55°C. After the specific gene is amplified by PCR, its specificity can be checked by agarose gel electrophoresis. The specific PCR results of the molecular sequence in other Helicobacter pylori are shown in Figure 4. Simultaneously, specific sequences can be obtained by Sanger sequencing of the PCR product, the specific sequence of Helicobacter pylori JMF0 is shown in SEQ.ID No.5, the specific sequence of Helicobacter pylori FMA6 is shown in SEQ.ID No.6, these specific The sequence also has the function of identifying standard strains.

表7幽门螺杆菌标准株的特异性靶标位点引物The specific target site primer of table 7 Helicobacter pylori standard strain

实施例8幽门螺杆菌标准株的定量检测方法The quantitative detection method of embodiment 8 Helicobacter pylori standard strain

本实施例提供了一种对样本中幽门螺杆菌标准株进行定量检测的方法，采用SEQ.ID No.7 及SEQ.ID No.8作为qPCR扩增反应的引物对幽门螺杆菌标准JFM0进行定量，采用SEQ.ID No.9 及SEQ.ID No.10作为qPCR扩增反应的引物对幽门螺杆菌标准FMA6进行定量，具体操作如下：This embodiment provides a method for quantitative detection of Helicobacter pylori standard strains in samples, using SEQ.ID No.7 and SEQ.ID No.8 as primers for qPCR amplification reactions to quantify Helicobacter pylori standard JFM0 , using SEQ.ID No.9 and SEQ.ID No.10 as primers for qPCR amplification reaction to quantify Helicobacter pylori standard FMA6, the specific operation is as follows:

(1)模板DNA制备(1) Template DNA preparation

提取含HP标准株的样本中的总微生物DNA，作为待检测模板。Extract the total microbial DNA in the sample containing the HP standard strain as the template to be detected.

(2)qPCR检测体系及扩增程序(2) qPCR detection system and amplification procedure

幽门螺杆菌标准株JMF0的qPCR反应体系配置如下：The qPCR reaction system configuration of Helicobacter pylori standard strain JMF0 is as follows:

幽门螺杆菌标准株FMA6的qPCR反应体系配置如下：The qPCR reaction system configuration of Helicobacter pylori standard strain FMA6 is as follows:

qPCR扩增程序：qPCR amplification program:

(3)qPCR结果读取(3) qPCR result reading

利用Roche荧光定量扩增仪上对体系进行扩增检测，利用软件SW 1.1读取扩增结果。在空白对照不存在荧光信号前提下，如果产生荧光信号，说明样品中含有对应的幽门螺杆菌标准菌株JMF0或FMA6；如果不产生荧光信号，则样品中不含有幽门螺杆菌标准菌株JMF0或FMA6。Using Roche The system was amplified and detected on the fluorescence quantitative amplification instrument, and the software was used to SW 1.1 reads the amplification result. Under the premise that there is no fluorescent signal in the blank control, if a fluorescent signal is generated, it means that the sample contains the corresponding Helicobacter pylori standard strain JMF0 or FMA6; if no fluorescent signal is generated, the sample does not contain the Helicobacter pylori standard strain JMF0 or FMA6.

(4)qPCR检测对幽门螺杆菌标准株的检测灵敏度评价(4) Evaluation of the detection sensitivity of qPCR detection to the standard strain of Helicobacter pylori

应用生理盐水将浓度为1×10⁹CFU/mL的HP标准株JMF0及FMA6按照10倍梯度进行稀释，得浓度为10⁹、10⁸、10⁷、10⁶、10⁵、10⁴、10³、10²、10¹CFU/mL的菌株纯培养物，参照说明书方法采用细菌基因组DNA提取试剂盒对不同浓度的幽门螺杆菌标准株进行基因组DNA 提取。按上述qPCR方案进行不同浓度的幽门螺杆菌进行定量检测，每个浓度样品分别进行三次平行实验。Use physiological saline to dilute the HP standard strains JMF0 and FMA6 with a concentration of 1×10 ⁹ CFU/mL according to a 10-fold gradient to obtain concentrations of 10 ⁹ , 10 ⁸ , 10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ , and 10 ³ , 10 ² , and 10 ¹ CFU/mL pure cultures of bacterial strains, and use bacterial genomic DNA extraction kits to extract genomic DNA from different concentrations of Helicobacter pylori standard strains according to the method in the manual. According to the above qPCR protocol, different concentrations of Helicobacter pylori were quantitatively detected, and three parallel experiments were carried out for each concentration sample.

绘制标准曲线：以样品中幽门螺杆菌浓度的对数为横坐标，以相应的qPCR的实时Ct值为纵坐标，拟合所得曲线即为HP定量检测的标准曲线。标准曲线如图5A所示，引物对样本中幽门螺杆菌标准株JMF0的检测限分别为1×10¹CFU/mL(图5A)，所述幽门螺杆菌标准株JMF0 的拟合标准曲线为y＝29.402-0.261x，相关系数R²为0.961(图5B)；引物对样本中幽门螺杆菌标准株FMA6的检测限分别为1×10²CFU/mL(图5C)，所述幽门螺杆菌标准株FMA6的拟合标准曲线为y＝28.893-0.253x，相关系数R²为0.928(图5D)。Draw a standard curve: take the logarithm of the concentration of Helicobacter pylori in the sample as the abscissa, and take the corresponding real-time Ct value of qPCR as the ordinate, and the fitted curve is the standard curve for HP quantitative detection. The standard curve is shown in Figure 5A, the detection limits of the primers for the Helicobacter pylori standard strain JMF0 in the sample are respectively 1×10 ¹ CFU/mL (Figure 5A), and the fitting standard curve of the Helicobacter pylori standard strain JMF0 is y =29.402-0.261x, the correlation coefficient R ² is 0.961 (Fig. 5B); the detection limit of the primers for the Helicobacter pylori standard strain FMA6 in the sample is respectively 1×10 ² CFU/mL (Fig. 5C), and the Helicobacter pylori standard strain The fitting standard curve of strain FMA6 was y=28.893-0.253x, and the correlation coefficient R ² was 0.928 (Fig. 5D).

图5是采用本发明提供的qPCR检测方案对含幽门螺杆菌标准株菌落数样品进行定量检测：A.结果提示当样本中幽门螺杆菌标准株JMF0菌落浓度≥1×10¹CFU/mL时，样本可检测出荧光信号；B.结果提示当样本中幽门螺杆菌标准株FMA6菌落浓度≥1×10²CFU/mL时，样本可检测出荧光信号；C.本发明对含幽门螺杆菌菌落JFM0样本的定量检测效果良好，标准曲线的拟合度高；D.本发明对含幽门螺杆菌菌落FMA6样本的定量检测效果良好，标准曲线的拟合度高。Fig. 5 is that the qPCR detection scheme provided by the present invention is used for quantitative detection of samples containing the number of colonies of Helicobacter pylori standard strains: A. The results suggest that when the concentration of Helicobacter pylori standard strain JMF0 colonies in the sample is ≥ 1×10 ¹ CFU/mL, The sample can detect a fluorescent signal; B. The results suggest that when the concentration of the Helicobacter pylori standard strain FMA6 colony in the sample is ≥ 1×10 ² CFU/mL, the sample can detect a fluorescent signal; The quantitative detection effect of the sample is good, and the fitting degree of the standard curve is high; D. The present invention has a good quantitative detection effect on the sample containing Helicobacter pylori colony FMA6, and the fitting degree of the standard curve is high.

(5)实际样本中幽门螺杆菌标准株qPCR定量检测(5) Quantitative detection of Helicobacter pylori standard strain qPCR in actual samples

采用幽门螺杆菌标准株JMF0或FMA6评估益生菌拮抗幽门螺杆菌的效能，将 1×10³CFU/mL幽门螺杆菌标准株JMF0及FMA6分别加入200μL幽门螺杆菌增殖培养基中，同时加入10μL待测的益生菌发酵上清，在37℃微需养环境培养72小时。72小时后收集幽门螺杆菌增殖培养基，按上述方法对增殖后的幽门螺杆菌标准株JMF0及FMA6进行定量。检测加入待测益生菌发酵液后，JMF0的Ct值由27.77±0.04升至28.23±0.06，带入计算公式可知加入待测益生菌后，幽门螺杆菌标准株JMF0的浓度由1×10^6.27CFU/mL降至1×10^4.49CFU/mL；检测加入待测益生菌发酵液后，FMA6的Ct值由27.38±0.03升至28.01±0.03，带入计算公式可知加入待测益生菌后，幽门螺杆菌标准株FMA6的浓度由1×10^6.00CFU/mL降至1×10^3.48CFU/mL。因此可知，待测益生菌具有同时拮抗我们不同生物型、不同毒力分型及MLST分型的幽门螺杆菌标准株 JMF0及FMA6。The Helicobacter pylori standard strain ^JMF0 or FMA6 was used to evaluate the anti-H. The probiotic fermentation supernatant tested was cultured in a microtrophic environment at 37°C for 72 hours. After 72 hours, the Helicobacter pylori proliferation medium was collected, and the proliferation of Helicobacter pylori standard strains JMFO and FMA6 were quantified according to the above method. After adding the probiotics to be tested, the Ct value of JMF0 increased from 27.77±0.04 to 28.23±0.06, and it was brought into the calculation formula to know that after adding the probiotics to be tested, the concentration of Helicobacter pylori standard strain JMF0 increased from 1×10 ^6.27 CFU /mL decreased to 1×10 ^4.49 CFU/mL; after adding the probiotics to be tested, the Ct value of FMA6 increased from 27.38±0.03 to 28.01±0.03. The concentration of the standard strain FMA6 decreased from 1×10 ^6.00 CFU/mL to 1×10 ^3.48 CFU/mL. Therefore, it can be seen that the probiotics to be tested can simultaneously antagonize our standard strains of Helicobacter pylori JMF0 and FMA6 of different biotypes, different virulence types and MLST types.

1.幽门螺杆菌JMF0的cagA氨基酸序列SEQ ID No.11. The cagA amino acid sequence of Helicobacter pylori JMF0 SEQ ID No.1

MTNETINQQPQTEAAFNPQQFINNLQVAFLKVDNAVASYDPDQKPIVDKNDRDNRQAFDGISQLREEYSNKAIKNPTKKN QYFSDFINKSNDLINKDNLIDVESSTKSFQKFGDQRYRIFTSWVSHQNDPSKINTRSIRNFMENIIQPPISDDKEKAEFL KSAKQSFAGIIIGNQIRTDQKFMGVFDESLKERQEAEKNGGPTGGDWLDIFLSFIFDKKQSSDVKEAINQEPVPHVQPDI ATTTTHIQGLPPESRDLLDERGNFSKFTLGDMEMLDVEGVADIDPNYKFNQLLIHNNALSSVLMGSHNGIEPEKVSLLYA GNGGFGAKHDWNATVGYKDQQGNNVATIINVHMKNGSGLVIAGGEKGINNPSFYLYKEDQLTGSQRALSQEEIRNKIDFM EFLAQNNAKLDDLSEKEKEKFRNEIKDFQKDSKAYLDALGNDRIAFVSKKDPKHSALITEFGNGDLSYTLKDYGKKADKA LDREKNVTLQGSLKHDGVMFIDYSNFKYTNASKSPDKGVGVTNGVSHLEAGFDKVAVFNLSNLNNLAITNPIRRDLEDKL TTKGLSPQEANKLIKDFLSSNKELVGKALNFNKAVAEAKNTGNYDEVKQAQKDLGKSLRKREHLEKDVAKNLESKSGNKN KMEAKSQANSQKDEIFALINKEANKDARAIAYTSNLKGIRRELSDKLENVNKNLKDFSKSFDEFKNGKNKDFSKAEETLK ALKGSVKDLGINPEWISKVENLNAALNEFKNGKNKDFSKVTQAKSDLENSVKDVIINQKVTDKVDNLSSAVSVAKATGDF SRVNQALADLKNFSKEQLAQQAQKNEDFNTGKKSEIYQSVKNGVNGTLVGNGLSKAEATTLSKNFSDIKKELNAKLGNFN NNNNGLKNEPIYAKVNKKKTGQAASLEEPIYAQVAKKVNAKIDRLNQIASGLGGVGQAAGFPLKKHDKVEDLSKVGLSAS PEPIYATIDDLGGPFPLKKHDKVEDLSKVGLSREQQLKQKIDNLNQAVSEAKAGFFGNLEQTIDNLKDSVKNNSVNLWVE RAKKVPASLSAKLDNYATNSHTRINSNIKNGAINEKATGMLMQKNPEWLKLVNDKIVAHNVGSVPLSEYDKIGFNQKNMKDYSDSFKFSTKLNNAVKDIKSGFTQFLTNAFSTGYYCLAGENAEHGIKNVNTKGGFQKS。MTNETINQQPQTEAAFNPQQFINNLQVAFLKVDNAVASYDPDQKPIVDKNDRDNRQAFDGISQLREEYSNKAIKNPTKKN QYFSDFINKSNDLINKDNLIDVESSTKSFQKFGDQRYRIFTSWVSHQNDPSKINTRSIRNFMENIIQPPISDDKEKAEFL KSAKQSFAGIIIGNQIRTDQ KFMGVFDESLKERQEAEKNGGPTGGDWLDIFLSFIFDKKQSSDVKEAINQEPVPHVQPDI ATTTTHIQGLPPESRDLLDERGNFSKFTLGDMEMLDVEGVADIDPNYKFNQLLIHNNALSSVLMGSHNGIEPEKVSLLYA GNGGFGAKHDWNATVGYKDQQGNNVATIINVHMKNGSGLVIAGGEKG INNPSFYLYKEDQLTGSQRALSQEEIRNKIDFM EFLAQNNAKLDDLSEKEKEKFRNEIKDFQKDSKAYLDALGNDRIAFVSKKDPKHSALITEFGNGDLSYTLKDYGKKADKA LDREKNVTLQGSLKHDGVMFIDYSNFKYTNASKSPDKGVGVTNGVSHLEAGFDKVAVFN LSNLNNLAITNPIRRDLEDKL TTKGLSPQEANKLIKDFLSSNKELVGKALNFNKAVAEAKNTGNYDEVKQAQKDLGKSLRKREHLEKDVAKNLESKSGNKN KMEAKSQANSQKDEIFALINKEANKDARAIAYTSNLKGIRRELSDKLENVNKNLKDFSKSFDEFKNGKNKD FSKAEETLK ALKGSVKDLGINPEWISKVENLNAALNEFKNGKNKDFSKVTQAKSDLENSVKDVIINQKVTDKVDNLSSAVSVAKATGDF SRVNQALADLKNFSKEQLAQQAQKNEDFNTGKKSEIYQSVKNGVNGTLVGNGLSKAEATTLSKNFSDIKKELNAKLGNFN NNNNGLK NEPIYAKVNKKKTGQAASLEEPIYAQVAKKVNAKIDRLNQIASGLGGVGQAAGFPLKKHDKVEDLSKVGLSAS PEPIYATIDDLGGPFPLKKHDKVEDLSKVGLSREQQLKQKIDNLNQAVSEAKAGFFGNLEQTIDNLKDSVKNNSVNLWVE RAKKVPASLSAKLDNYATNSHTRINSNI KNGAINEKATGMLMQKNPEWLKLVNDKIVAHNVGSVPLSEYDKIGFNQKNMKDYSDSFKFSTKLNNAVKDIKSGFTQFLTNAFSTGYYCLAGENAEHGIKNVNTKGGFQKS.

2.幽门螺杆菌FMA6的cagA氨基酸序列SEQ.ID No.22. The amino acid sequence of cagA of Helicobacter pylori FMA6 SEQ.ID No.2

MTNETIDQTITPDQTPNQTDFVPQRFINNLQVAFIKVGGAVASFDPDQKPIVDKNDRDNRQAFEKISQLREEYANKAIKN PAKKNQYFSDFINKSNDLINKDNLIAVDSSVESFKKFGDQRYQIFTSWVSLQKDPSKINTQQIRNFMENIIQPPISDDKE KAEFLRSAKQSFAGIIIGNQIRSDQKFMGVFDESLKERQEAEKNAEPSGGDWLDIFLSFVFNKKQSSDLKETLNQEPRPD FEQNLATTTTDIQGLPPEARDLLDERGNFFKFTLGDVEMLDVEGVADKDPNYKFNQLLIHNNALSSVLMGGHSNIEPEKV SLLYGDNGGPEARHDWNATVGYKNQQGNNVATLINAHLNNGSGLIIAGNEDGIKNPSFYLYKEDQLTGLKQAMSQEEIQN KVDFMEFLAKNNAKLDNLSEKEKEKFQTEIENFQKDRKAYLDALGNDHIAFVSKKDPKHLALVTEFGNGEVSYTLKDYGK KQDKALDGETKTTLQGNLKYDGVMFVNYSNFKYTNASKSPDKGVGATNGVSHLEANFSKVAVFNLPNLNNLAITNYIRRD LEDKLLAEGLSPQEANKLIKDFLNSNKQLVGKISNFNKAVAEAKNTGNYDEVKKAQKDLEKSLRKREHLEKEVAKKLESR NDNKNRMEAKAQANSQKDKIFALINKEASKEARAAAFDPNLKGVRIELSDKLENINKNLKDFGKSFDELKNGKNNDFSKA EETLKALKDSVKDLGINPEWISKIENLNAALNDFKNGKNNDFSKVTQAKSDLENSIKDVHINQKITNKANDLNQAVLETK LTGDFSKVEQALAELKSLSLDLGKNSDLQKSVKNGVNGTLVGNGLSKTEATTLTKNFSDIRKELNEKVFGNSNNNNNGLK NNTEPIYAQVNKKKTGQVASPEEPIYAQVAKKVSAKIDQLNEATSAINRKIDRINKIASAGKGVGGFSGAGQSASPEPIY ATIDFDEANQAGFPLRRSAAVNDLSKVGLSREQELTRRIGDLNQAVSEAKTGRFDKLEQKIDELKDSTKKNALKLWVESA KQVPTSLQAKLDNYATNSHTRINSNVQDGTINEKATGMLTQKNPEWLKLVNDKIVAHNVGSAHLSEYDKIEFNQKNMKDYSDSFKFSTKLNNAVKDIKSSFVQFLTNTFSTGSYSLMKENAEHGVKNTNTKGGFQKS。MTNETIDQTITPDQTPNQTDFVPQRFINNLQVAFIKVGGAVASFDPDQKPIVDKNDRDNRQAFEKISQLREEYANKAIKN PAKKNQYFSDFINKSNDLINKDNLIAVDSSVESFKKFGDQRYQIFTSWVSLQKDPSKINTQQIRNFMENIIQPPISDDKE KAEFLRSAKQSFAGIIIG NQIRSDQKFMGVFDESLKERQEAEKNAEPSGGDWLDIFLSFVFNKKQSSDLKETLNQEPRPD FEQNLATTTTDIQGLPPEARDLLDERGNFFKFTLGDVEMLDVEGVADKDPNYKFNQLLIHNNALSSVLMGGHSNIEPEKV SLLYGDNGGPEARHDWNATVGYKNQQGNNVATLINA HLNNGSGLIIAGNEDGIKNPSFYLYKEDQLTGLKQAMSQEEIQN KVDFMEFLAKNNAKLDNLSEKEKEKFQTEIENFQKDRKAYLDALGNDHIAFVSKKDPKHLALVTEFGNGEVSYTLKDYGKKQDKALDGETKTTLQGNLKYDGVMFVNYSNFKYTNASKSPDKG VGATNGVSHLEANFSKVAVFNLPNLNNLAITNYIRRD LEDKLLAEGLSPQEANKLIKDFLNSNKQLVGKISNFNKAVAEAKNTGNYDEVKKAQKDLEKSLRKREHLEKEVAKKLESRNDNKNRMEAKAQANSQKDKIFALINKEASKEARAAAFDPNLKGVRIELELENINKNLKDFG KSFDELKNGKNNDFSKA EETLKALKDSVKDLGINPEWISKIENLNAALNDFKNGKNNDFSKVTQAKSDLENSIKDVHINQKITNKANDLNQAVLETK LTGDFSKVEQALAELKSLSLLDLGKNSDLQKSVKNGVNGTLVGNGLSKTEATTLTKNFSDIRKELNEKVFGNSNNNNNGLK NNTEPIYAQVNKKKTGQVASPEEPIYAQVAKKVSAKIDQLNEATSAINRKIDRINKIASAGKGVGGFSGAGQSASPEPIYATIDFDEANQAGFPLRRSAAVNDLSKVGLSREQELTRRIGDLNQAVSEAKTGRFDKLEQKIDELKDSTKKNALKLWVESA KQVPTSLQAKLDNYATNSH TRINSNVQDGTINEKATGMLTQKNPEWLKLVNDKIVAHNVGSAHLSEYDKIEFNQKNMKDYSDSFKFSTKLNNAVKDIKSSFVQFLTNTFSTGSYSLMKENAEHGVKNTNTKGGFQKS.

3.幽门螺杆菌JMF0的特异基因SEQ.ID No.33. The specific gene SEQ.ID No.3 of Helicobacter pylori JMF0

ATGATTGATGTGGCGTATCTGTGCGTTAAGGAGACTTCTTCTAGGGTTGAAGAAAGAGAGAAAGAATTATTAAGCCTTAC TAAAGAATGGAATATCCCAACGATTGTCATCTTCACCAACACCCAAGCCGAAGCTGGCGATGCGTTTGTTCAAGAATCTC AAAGAGTTATAGATGAAGAATGGGGCTTTAAGGGTTTTATCAGAGCCTATGCGAGAGTCAATTCCGTTAAATATTCATTT AGAGGCATGGAAGTCCCTGTTGAAGGTTTAAAAGAATTGGTAGATGAAACGAAAAAATGTCTTTCAGACGCTATGAGAAA CCATTTTTTGCGTGTTCAAAAAATTAAGATTCAAGAAAGAAAACAAGCTATGATAGATGAAAGTAAATCCATTATCCACT TTGCATCAGGAGCGGCTGGGGTTAGCCCTATACCCCTTAGCGATATGCCCCTTATTACAACTGCTCAGATGGCAATGATT TATAAAATGAATAGGGCGTTTAAAGTGAAAATGGAAGAATCCATAGTCGCATCGCTAATCACCGGATTGTTAGGTTTTAC CGCTATTGGACAAACAGCAAAAACGATTGTTGCTAATTTGATCAAATGCATTCCTGGTGTTGGGAGTGTTGTGGGGGCAC GACCGCTGCGGTTATCACAGAAGGCATTGGGTTTGCTTATTTGA。ATGATTGATGTGGCGTATCTGTGCGTTAAGGAGACTTCTTCTAGGGTTGAAGAAAGAGAGAAAGAATTATTAAGCCTTAC TAAAGAATGGAATATCCCAACGATTGTCATTCTTCACCAACACCCAAGCCGAAGCTGGCGATGCGTTTGTTTATAGATGAAGAATGGGGCTTTAAGGGTTTTATCAGAGCCTATGCGAGAGTCAATTC CGTTAAATATTCATTT AGAGGCATGGAAGTCCCTGTTGAAGGTTTAAAAAGAATTGGTAGATGAAACGAAAAAATGTCTTTCAGACGCTATGAGAAACCATTTTTTGCGTGTTAAAAAATTAAGATTCAAGAAAGAAAACAAGCTATGATAGATGAAAGTAAATCCATTATCACT TTGCATCAGGAGCGGCTGGGGTTAGCCCTATACCCCTTAGCGATAT GCCCCTTATTACAACTGCTCAGATGGCAATGATT TATAAAATGAATAGGGCGTTTAAAGTGAAAATGGAAGAATCCATAGTCGCATCGCTAATCACCGGATTGTTAGGTTTTAC CGCTATTGGACAAACAGCAAAAACGATTGTTGCTAATTTGATCAAATGCATTCCTGGTGTTGGGAGTGTTGTGGGGGCAC GACCGCTGCGGTTATCACAGAAG GCATTGGGTTTGCTTATTTGA.

4.幽门螺杆菌FMA6的特异基因SEQ.ID No.44. Specific gene SEQ.ID No.4 of Helicobacter pylori FMA6

ATGAATTACCATAACCTACCTAATAGCACTTTAGAAATAAGCAAACAACCACAGGTCAAAGAAATCACTAACGAATTATT AAAGCAACTACAGAACGCACTAAATTCTAATAGTCTTTTTACCGAGCAAGTGGAACTGAGCCTTAAAGGTATCGTTAGGA TTTTAGAAGTGCTTTTGAGTTTGGATTTTTTTAAAAACACCAATGAGATTGATACCAACTTAAGAAATTCCATTGAATGG CTTAATAACGCCGGCGAGAGCTTGAAAATTAAAATGAAAGAATACGAGAGCTTTTTTACTGATTTCAATACGAGCATGCG CGCTAACGAGCAAGAAGTAACCAACACCTTAAACGCTAACACCGAAAACATTTTTATTTAA。ATGAATTACCATAACCTACCTAATAGCACTTTAGAAATAAGCAAACAACCACAGGTCAAAGAAATCACTAACGAATTATT AAAGCAACTACAGAACGCACTAAATTCTAATAGTCTTTTTACCGAGCAAGTGGAACTGAGCCTTAAAGGTATCGTTAGGA TTTTAGAAGTGCTTTTGAGTTTGGATTTTTTTAAAAAACACCAATGAGATTGATACCAACTTAAGAAATTC CATTGAATGG CTTAATAACGCCGGCGAGAGCTTGAAAATTAAAATGAAAGAATACGAGAGCTTTTTTACTGATTTCAATACGAGCATGCG CGCTAACGAGCAAGAAGTAACCAACACCTTAAACGCTAACACCGAAAACATTTTTATTTAA.

5.幽门螺杆菌JMF0的特异序列SEQ.ID No.55. The specific sequence SEQ.ID No.5 of Helicobacter pylori JMF0

GGCATGGAAGTCCCTGTTGAAGGTTTAAAAGAATTGGTAGATGAAACGAAAAAATGTCTTTCAGACGCTATGAGAAACCA TTTTTTGCGTGTTCAAAAAATTAAGATTCAAGAAAGAAAACAAGCTATGATAGATGAAAGTAAATCCATTATCCACTTTG CATCAGGAGCGGCTGGGGTTAGCCCTATACCCCTTAGCGATATGCCCCTTATTACAACTGCTCAGATGGCAATGATTTAT AAAATGAATAGGGCGTTTAAAGTGAAAATGGAAGAATCCATAGTCGCATCGCTAATCACCGGATTGTTAGGTTTTACCGC TATTGGACAAACAGCAAAAACGATTGTTGCTAATTTGATCAAATGCATTCCTGGTGTTGGGAGTGTTG。GGCATGGAAGTCCCTGTTGAAGGTTTAAAAAGAATTGGTAGATGAAACGAAAAAATGTCTTTCAGACGCTATGAGAAACCA TTTTTTGCGTGTTCAAAAAATTAAGATTCAAAAAGAAAACAAGCTATGATAGATGAAAGTAAATCCATTATCCACTTTG CATCAGGAGCGGCTGGGGTTAGCCCTATACCCCTTAGCGATATGCCCCTTATTACACTGC TCAGATGGCAATGATTTAT AAAATGAATAGGGCGTTTAAAGTGAAAATGGAAGAATCCATAGTCGCATCGCTAATCACCGGATTGTTAGGTTTTTACCGC TATTGGACAAACAGCAAAAACGATTGTTGCTAATTTGATCAAATGCATTCCTGGTGTTGGGAGTGTTG.

6.幽门螺杆菌FMA6的特异序列SEQ.ID No.66. The specific sequence SEQ.ID No.6 of Helicobacter pylori FMA6

ACCGAGCAAGTGGAACTGAGCCTTAAAGGTATCGTTAGGATTTTAGAAGTGCTTTTGAGTTTGGATTTTTTTAAAAACAC CAATGAGATTGATACCAACTTAAGAAATTCCATTGAATGGCTTAATAACGCCGGCGAGAGCTTGAAAATTAAAATGAAAG AATACGAGAGCTTTTTTACTGATTTCAATACGAGCATGCGCG。ACCGAGCAAGTGGAACTGAGCCTTAAAGGTATCGTTAGGATTTTAGAAGTGCTTTTGAGTTTGGATTTTTTTAAAAACAC CAATGAGATTGATACCAACTTAAGAAATTCCATTGAATGGCTTAATAACGCCGGCGAGAGCTTGAAAATTAAAATGAAAG AATACGAGAGCTTTTACTGATTTCAATACGAGCATGCGCG.

7.幽门螺杆菌JMF0的特异序列SEQ.ID No.5的检测上游引物SEQ.ID No.77. The detection upstream primer SEQ.ID No.7 of the specific sequence SEQ.ID No.5 of Helicobacter pylori JMF0

GGCATGGAAGTCCCTGTTGA。GGCATGGAAGTCCCTGTTGA.

8.幽门螺杆菌JMF0的特异序列SEQ.ID No.5的检测下游引物SEQ.ID No.88. Detection of the specific sequence SEQ.ID No.5 of Helicobacter pylori JMF0 downstream primer SEQ.ID No.8

CAACACTCCCAACACCAGGA。CAACACTCCCAACACCAGGA.

9.幽门螺杆菌FMA6的特异序列SEQ.ID No.6的检测上游引物SEQ.ID No.99. The detection upstream primer SEQ.ID No.9 of the specific sequence SEQ.ID No.6 of Helicobacter pylori FMA6

ACCGAGCAAGTGGAACTGAG。ACCGAGCAAGTGGAACTGAG.

10.幽门螺杆菌FMA6的特异序列SEQ.ID No.6的检测下游引物SEQ.ID No.1010. Detection of the specific sequence SEQ.ID No.6 of Helicobacter pylori FMA6 downstream primer SEQ.ID No.10

CGCGCATGCTCGTATTGAAA。CGCGCATGCTCGTATTGAAA.

11、幽门螺杆菌JMF0的16S rRNA基因序列SEQ ID No.1111. The 16S rRNA gene sequence of Helicobacter pylori JMF0 SEQ ID No.11

AGAGTTTGATCCTGGCTCAGAGTGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGA ACGATGAAGCTTCTAGCTTGCTAGAAGGCTGATTAGTGGCGCACGGGTGAGTAACGCA TAGGTCATGTGCCTCTTAGTTTGGGATAGCCATTGGAAACGATGATTAATACCAGATAC TCCCTACGGGGGAAAGATTTATCGCTAAGAGATCAGCCTATGTCCTATCAGCTTGTTGG TAAGGTAATGGCTTACCAAGGCTATGACGGGTATCCGGCCTGAGAGGGTGAACGGACACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATATTGCT CAATGGGGGAAACCCTGAAGCAGCAACGCCGCGTGGAGGATGAAGGTTTTAGGATTGT AAACTCCTTTTGTTAGAGAAGATAATGACGGTATCTAACGAATAAGCACCGGCTAACT CCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTACTCGGAATCACTGGGCG TAAAGAGCGCGTAGGCGGGATAGTCAGTCAGGTGTGAAATCCTATGGCTTAACCATAG AACTGCATTTGAAACTACTATTCTAGAGTGTGGGAGAGGTAGGTGGAATTCTTGGTGT AGGGGTAAAATCCGTAGAGATCAAGAGGAATACTCATTGCGAAGGCGACCTGCTGGA ACATTACTGACGCTGATTGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGG TAGTCCACGCCCTAAACGATGGATGCTAGTTGTTGGAGGGATTAGTCTTTCCAGTAATG CAGCTAACGCATTAAGCATCCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAG GAATAGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGATACACG AAGAACCTTACCTAGGCTTGACATTGAGAGAATCCGCTAGAAATAGTGGAGTGTCTGG CTTGCCAGACCTTGAAAACAGGTGCTGCACGGCTGTCGTCAGCTCGTGTCGTGAGATGT TGGGTTAAGTCCCGCAACGAGCGCAACCCCCTTTCTTAGTTGCTAACAGGTCATGCTGA GAACTCTAAGGATACTGCCTCCGTAAGGAGGAGGAAGGTGGGGACGACGTCAAGTCA TCATGGCCCTTACGCCTAGGGCTACACACGTGCTACAATGGGGTGCACAAAGAGAAGC AATACTGCGAAGTGGAGCCAATCTTCAAAACACCTCTCAGTTCGGATTGTAGGCTGCA ACTCGCCTGCATGAAGCTGGAATCGCTAGTAATCGCAAATCAGCCATGTTGCGGTGAA TACGTTCCCGGGTCTTGTACTCACCGCCCGTCACACCATGGGAGTTGTGTTTGCCTTAA GTCAGGATGCTAAATTGGCTACTGCCCACGGCACACACAGCGACTGGGGTGAAGTCGT AACAAGGTAGCCGTAG。AGAGTTTGATCCTGGCTCAGAGTGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGA ACGATGAAGCTTCTAGCTTGCTAGAAGGCTGATTAGTGGCGCACGGGTGAGTAACGCA TAGGTCATGTGCCTCTTAGTTTGGGATAGCCATTGGAAACGATGATTAATACCAGATAC TCCCTACGGGGGAAAGATTTATCGCTAAGAGATCAGCCT ATGTCCTATCAGCTTGTTGG TAAGGTAATGGCTTACCAAGGCTATGACGGGTATCCGGCCTGAGAGGGTGAACGGACACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATATTGCT CAATGGGGGAAACCCTGAAGCAGCAACGCCGCGTGGAGGATGAAGGTTTTAGGATTGT AAACTCCTTTTGT TAGAGAAGATAATGACGGTATCTAACGAATAAGCACCGGCTAACT CCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTACTCGGAATCACTGGGCG TAAAGAGCGCGTAGGCGGGATAGTCAGTCAGGTGTGAAATCCTATGGCTTAACCATAG AACTGCATTTGAAACTACTATTCTAGAGTGTGGGAGAGGTAGGTGGAATTCTT GGTGT AGGGGTAAAATCCGTAGAGATCAAGAGGAATACTCATTGCGAAGGCGACCTGCTGGA ACATTACTGACGCTGATTGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGG TAGTCCACGCCCTAAACGATGGATGCTAGTTGTTGGAGGGATTAGTCTTTCCAGTAATG CAGCTAACGCATTAAGCATCCCGCCTGGG GAGTACGGTCGCAAGATTAAAACTCAAAG GAATAGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGATACACG AAGAACCTTACCTAGGCTTGACATTGAGAGAATCCGCTAGAAATAGTGGAGTGTCTGG CTTGCCAGACCTTGAAAACAGGTGCTGCACGGCTGTCGTCAGCTCGTGTCGTGAGATGT TGGGTTAAGTCCC GCAACGAGCGCAACCCCCTTTCTTAGTTGCTAACAGGTCATGCTGA GAACTCTAAGGATACTGCCTCCGTAAGGAGGAGGAAGGTGGGGACGACGTCAAGTCA TCATGGCCCTTACGCCTAGGCTACACACGTGCTACAATGGGGTGCACAAAGAGAAGC AATACTGCGAAGTGGAGCCAATCTTCAAAAACACCTTCAGTTCGGATTGTAGGCT GCA ACTCGCCTGCATGAAGCTGGAATCGCTAGTAATCGCAATCAGCCATGTTGCGGTGAA TACGTTCCCGGGTCTTGTACTCACCGCCCGTCACACCATGGGAGTTGTGTTTGCCTTAA GTCAGGATGCTAAATTGGCTACTGCCCACGGCACACACAGCGACTGGGGTGAAGTCGT AACAAGGTAGCCGTAG.

12、幽门螺杆菌FMA6的16S rRNA基因序列SEQ ID No.1212. The 16S rRNA gene sequence of Helicobacter pylori FMA6 SEQ ID No.12

AGAGTTTGATCCTGGCTCAGAGTGAACGCTGGCGGCGTGCCTAATACATGCAAGT CGAACGATGAAGCTTTCTAGCTTGCTAGAAGGCTGATTAGTGGCGCACGGGTGAGTAA CGCATAGGTCATGTGCCTCTTAGTTTGGGATAGCCATTGGAAACGATGATTAATACCAG ATACTCCCTACGGGGGAAAGATTTATCGCTAAGAGATCAGCCTATGTCCTATCAGCTTG TTGGTAAGGTAATGGCTTACCAAGGCTATGACGGGTATCCGGCCTGAGAGGGTGAACG GACACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATA TTGCTCAATGGGGGAAACCCTGAAGCAGCAACGCCGCGTGGAGGATGAAGGTTTTAGG ATTGTAAACTCCTTTTGTTAGAGAAGATAATGACGGTATCTAACGAATAAGCACCGGC TAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTACTCGGAATCACT GGGCGTAAAGAGCGCGTAGGCGGGGTAGTCAGTCAGGTGTGAAATCCTATGGCTTAAC CATAGAACTGCATTTGAAACTACTATTCTAGAGTGTGGGAGAGGTAGGTGGAATTCTT GGTGTAGGGGTAAAATCCGTAGAGATCAAGAGGAATACTCATTGCGAAGGCGACCTGC TGGAACATTACTGACGCTGATTGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACC CTGGTAGTCCACGCCCTAAACGATGGATGCTAGTTGTTGGAGGGCTTAGTCTCTCCAGT AATGCAGCTAACGCATTAAGCATCCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTC AAAGGAATAGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGATA CACGAAGAACCTTACCTAGGCTTGACATTGAGAGAATCCGCTAGAAATAGTGGAGTGT CTGGCTTGCCAGACCTTGAAAACAGGTGCTGCACGGCTGTCGTCAGCTCGTGTCGTGA GATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCCTTTCTTAGTTGCTAACAGGTCAT GCTGAGAACTCTAAGGATACTGCCTCCGTAAGGAGGAGGAAGGTGGGGACGACGTCA AGTCATCATGGCCCTTACGCCTAGGGCTACACACGTGCTACAATGGGGTGCACAAAGA GAAGCAATACTGCGAAGTGGAGCCAATCTTCAAAATACCTCTCAGTTCGGATTGTAGG CTGCAACTCGCCTGCATGAAGCTGGAATCGCTAGTAATCGCAAATCAGCCATGTTGCG GTGAATACGTTCCCGGGTCTTGTACTCACCGCCCGTCACACCATGGGAGTTGTGTTTGC CTTAAGTCAGGATGCTAAATTGGCTACTGCCCACGGCACACACAGCGACTGGGGTGAA GTCGTAACAAGGTAGCCGTAG。AGAGTTTGATCCTGGCTCAGAGTGAACGCTGGCGGCGTGCCTAATACATGCAAGT CGAACGATGAAGCTTTTCTAGCTTGCTAGAAGGCTGATTAGTGGCGCACGGGTGAGTAA CGCATAGGTCATGTGCCTCTTAGTTTGGGATAGCCATTGGAAACGATGATTAATACCAG ATACTCCCTACGGGGGAAAGATTTATCGCTAAGAGATCAGCCTAT GTCCTATCAGCTTGTTGGTAAGGTAATGGCTTACCAAGGCTATGACGGGTATCCGGCCTGAGAGGGTGAACGGACACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATATTGCTCAATGGGGGAAACCCTGAAGCAGCAACGCCGCGTGGAGGATGAAGGTTTTAGG ATTGTAAACTCCTTTT GTTAGAGAAGATAATGACGGTATCTAACGAATAAGCACCGGC TAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTACTCGGAATCACT GGGCGTAAAGAGCGCGTAGGCGGGGTAGTCAGTCAGGTGTGAAATCCTATGGCTTAAC CATAGAACTGCATTTGAAACTACTATTCTAGAGTGTGGGAGAGGTAGGTGGAATTCTT G GTGTAGGGGTAAAATCCGTAGAGATCAAGAGGAATACTCATTGCGAAGGCGACCTGC TGGAACATTACTGACGCTGATTGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACC CTGGTAGTCCACGCCCTAAACGATGGATGCTAGTTGTTGGAGGGCTTAGTCTCTCCAGTAATGCAGCTAACGCATTAAGCATCCCGCCTGGGAGTACG GTCGCAAGATTAAAACTC AAAGGAATAGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGATA CACGAAGAACCTTACCTAGGCTTGACATTGAGAGAATCCGCTAGAAATAGTGGAGTGT CTGGCTTGCCAGACCTTGAAAACAGGTGCTGCACGGCTGTCGTCAGCTCGTGTCGTGA GATGTTGGGTTAAGTCCCGCAACGAGC GCAACCCCCTTTCTTAGTTGCTAACAGGTCAT GCTGAGAACTCTAAGGATACTGCCTCCGTAAGGAGGAGGAAGGTGGGGACGACGTCA AGTCATCATGGCCCTTACGCCTAGGGCTACACACGTGCTACAATGGGGTGCACAAAGA GAAGCAATACTGCGAAGTGGAGCCAATCTTCAAAATACCTCTCAGTTCGGATTGTAGG CTGCAACTCGC CTGCATGAAGCTGGAATCGCTAGTAATCGCAAATCAGCCATGTTGCG GTGAATACGTTCCCGGGTCTTGTACTCACCGCCCGTCACACCATGGGAGTTGTGTTTGC CTTAAGTCAGGATGCTAAATTGGCTACTGCCCACGCACACAGCGACTGGGGTGAA GTCGTAACAAGGTAGCCGTAG.

以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。The above-mentioned embodiments only express several implementation modes of the present invention, and its description is relatively specific and detailed, but it should not be interpreted as limiting the patent scope of the invention. It should be noted that for those skilled in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention. Therefore, the scope of protection of the patent for the present invention should be based on the appended claims.

Claims (2)

1. Application of primer group for detecting molecular target of helicobacter pylori in preparing helicobacter pylori strain detection reagent;

the molecular target is any nucleotide sequence shown as SEQ ID NO. 3-6; wherein the primer group for detecting the molecular targets described by SEQ ID NO.3 and SEQ ID NO.5 is used for preparing a detection reagent for detecting helicobacter pylori JMF0; the primer group for detecting the molecular targets described by SEQ ID No.4 and SEQ ID No.6 is used for preparing a detection reagent for detecting helicobacter pylori FMA6, wherein helicobacter pylori is [ ]Helicobacter pylori) JMF0, accession No. GDMCC No:62496 helicobacter pyloriHelicobacter pylori) FMA6, accession No. GDMCC No:62494.

2. the use according to claim 1, wherein the PCR primer for amplification against the nucleotide sequence shown as SEQ ID No.5 comprises: an upstream primer shown as SEQ ID NO.7 and a downstream primer shown as SEQ ID NO.8; the PCR primers for amplification against the nucleotide sequence shown as SEQ ID NO.6 include: an upstream primer shown as SEQ ID NO.9 and a downstream primer shown as SEQ ID NO.10.

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CN116064273B - Helicobacter pylori standard strain JMF0 and FMA6 containing specific molecular targets, detection and application thereof - Google Patents

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