WO2020168950A1 - 大肠杆菌o157:h7的cpa引物及试剂盒和检测方法 - Google Patents

大肠杆菌o157:h7的cpa引物及试剂盒和检测方法 Download PDF

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WO2020168950A1
WO2020168950A1 PCT/CN2020/074822 CN2020074822W WO2020168950A1 WO 2020168950 A1 WO2020168950 A1 WO 2020168950A1 CN 2020074822 W CN2020074822 W CN 2020074822W WO 2020168950 A1 WO2020168950 A1 WO 2020168950A1
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primer
target
seq
cross
stx1
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徐振波
骆玉婷
刘君彦
张桂兰
苗健
袁牧
陈玲
苏健裕
李冰
李琳
李晓玺
张霞
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South China University of Technology SCUT
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a CPA primer of Escherichia coli O157:H7, a detection kit and a detection method thereof.
  • microorganism detection and identification methods are mainly divided into culture identification method, immunoassay method and nucleic acid detection.
  • the cultivation identification method and the immunoassay method are cumbersome to operate, the experiment cycle is long, and the professional level of the experiment personnel is high.
  • Crossing Priming Amplification Compared with other nucleic acid amplification technologies, Crossing Priming Amplification (CPA) technology can quickly, efficiently, and specifically amplify target sequences under isothermal conditions. It is easy to operate, does not require precise temperature changing equipment, and costs Low, showing broad development prospects in the field of food-borne microbial detection. However, the design of primers for this reaction is more difficult. It is necessary to design five primers in a limited product length, and to avoid non-specific amplification of the five primers themselves and affect the results, so the design of primers is particularly important.
  • Escherichia coli O157:H7 is the most important serotype of enterohemorrhagic Escherichia coli. It is a zoonotic enteric pathogen, which infects humans mainly through contaminated animal-derived food, such as raw or minced meat products, Raw milk and contaminated vegetables and sprouts are transmitted to humans, and the corresponding clinical symptoms appear after infecting humans. Mild infections can cause abdominal cramps and diarrhea. Fever and vomiting may also occur. Severe infections are manifested as hemorrhagic colitis and hemolytic uremic syndrome.
  • E.coli O157:H7 there are some Escherichia coli that can produce Shiga toxin type I. If infection occurs, it can cause some gastrointestinal diseases such as diarrhea and hemorrhagic colitis in humans. After Shiga toxin type I is produced in the intestinal cavity, it can pass through epithelial cells and enter the blood circulation, causing vascular damage to specific target tissues, such as brain and kidney vascular damage. Shiga toxin-producing Escherichia coli has the characteristics of strong pathogenicity and lethality, which seriously threatens human life and health and has become a worldwide public health problem.
  • Chinese patent application CN 108796098A discloses a primer, kit and method for detecting E. coli Shiga toxin by PSR isothermal amplification reaction. This patent designs a PSR reaction primer for the detection of the specific target sequence stx2 of Shiga toxin.
  • Chinese patent application CN 108796099A discloses a PSR detection primer, kit and detection method for Escherichia coli O157:H7. This patent designs a PSR reaction primer for the specific target sequence rfbE of Escherichia coli O157:H7 for detection.
  • the minimum unit of reaction sensitivity in the above patent is pg/ ⁇ L, and the sensitivity is not high enough, which limits the scope of application.
  • the primary purpose of the present invention is a CPA primer of Escherichia coli O157:H7, the sensitivity of which reaches fg/ ⁇ L, which is 100-1000 times that of the PSR reaction, and can solve the prior art The problem of low sensitivity.
  • Another object of the present invention is to provide a CPA detection kit for Escherichia coli O157:H7.
  • Another object of the present invention is to provide a CPA detection method for Escherichia coli O157:H7, which has the characteristics of high sensitivity, good specificity, simple and rapid operation, accurate and reliable results, low detection cost, and suitable for field detection applications.
  • a CPA detection primer for Escherichia coli O157:H7 designed for its target rfbE, including stripping primers 4s and 5a, cross-amplifying primers 2a1s, and specific primers 2a, 3a; their nucleotide sequences are as follows: :
  • Target rfbE stripping primer 4s 5’-aggaccgcagaggaaaga-3’ (SEQ ID NO.1);
  • Target rfbE stripping primer 5a 5’-tccacgccaaccaagatc-3’ (SEQ ID NO. 2);
  • Target rfbE cross primer 2a1s 5’-agtacattggcatcgtgtcagataaactcatcgaaaca-3’ (SEQ ID NO.3);
  • Target rfbE specific primer 2a 5’-agtacattggcatcgtgt-3’ (SEQ ID NO.4);
  • Target rfbE specific primer 3a 5'-ggcatcgtgtggacagggt-3' (SEQ ID NO. 5).
  • a CPA detection primer for Escherichia coli Shiga toxin type I is designed for its target stx1, including stripping primers 4s and 5a, cross-amplification primer 2a1s, and specific primers 2a, 3a; its nucleotide sequences are as follows Shown:
  • Target stx1 stripping primer 4s 5’-agttgatgtcagagggatag-3’ (SEQ ID NO.6);
  • Target stx1 stripping primer 5a 5’-cgctgttgtacctggaaa-3’ (SEQ ID NO. 7);
  • Target stx1 cross primer 2a1s 5’-atcagcaaagcgataaaactacggcttattgttgaa-3’ (SEQ ID NO.8);
  • Target stx1 specific primer 2a 5’-atcagcaaagcgataaaa-3’ (SEQ ID NO.9);
  • Target stx1 specific primer 3a 5'-cctgttaacaaatcctgtcac-3' (SEQ ID NO. 10).
  • a CPA detection kit for Escherichia coli O157:H7 comprising the above-mentioned CPA detection primer for Escherichia coli O157:H7 and/or the CPA detection primer for Escherichia coli Shiga toxin type I;
  • the concentration of each CPA detection primer is preferably 10 ⁇ M
  • the kit also includes the following components:
  • reaction buffer 40.0mM Tris-HCl, 20.0mM ammonium sulfate, 20.0mM potassium chloride, 16.0mM magnesium sulfate, 0.2%(v/v) Tween 20, 1.4M betaine , 10.0mM dNTPs(each);
  • the Bst DNA polymerase described in component B is preferably an aqueous solution of Bst DNA polymerase with a concentration of 8 U/ ⁇ L.
  • the mixed solution of calcein and manganese chloride described in component C is prepared by the following method:
  • a method for detecting CPA of Escherichia coli O157:H7 including the following steps:
  • the cross primer constant temperature amplification reaction system is: 2 ⁇ reaction buffer 12.5 ⁇ L, 10 ⁇ M stripping primer 4s and 10 ⁇ M stripping primer 5a each 1.5 ⁇ L, 10 ⁇ M cross primer 2a1s 2.5 ⁇ L, 10 ⁇ M specific primer 2a and 10 ⁇ M Specific primers 3a are each 1.25 ⁇ L, DNA template 1.0 ⁇ L, 8U/ ⁇ L Bst DNA polymerase 1.0 ⁇ L, add nucleic acid water to make up to 25 ⁇ L; finally add 1 ⁇ L of a mixed solution of calcein and manganese chloride;
  • the present invention has the following advantages and effects:
  • the cross-primer isothermal amplification reaction detection and identification system designed for the E. coliO157:H7 specific target sequence rfbE and stx1 provided in the present invention solves the long cycle, low sensitivity and high cost required by the prior art method , Defects such as difficulty in field application.
  • a pair of stripping primers, cross primers and specific primers are designed to construct a cross primer constant temperature amplification reaction system, and the test results are obtained in about 60 minutes to shorten the traditional E. coli detection cycle.
  • the present invention is amplified under constant temperature conditions without time loss due to temperature changes, and it takes a short time.
  • the technology does not require special and expensive instruments and reagents, and the amplified products do not require gel electrophoresis. Direct color development with fluorescent dyes can judge the result by naked eyes, the operation is simple and fast, and the detection cost is low.
  • the kit and method of the present invention are particularly suitable for small and medium-sized units and on-site detection.
  • Figure 1 shows the gel electrophoresis results and color results of E. coli O157:H7 detected by cross-primer isothermal amplification reaction technology
  • Figure A shows the gel electrophoresis results of E. coli O157:H7 detected by cross-primer isothermal amplification
  • lane 1 is Escherichia coli O157:H7 ATCC43895
  • lane 2 is Escherichia coli O157:H7 ATCC43894
  • lane 3 is Escherichia coli O157:H7 E019
  • lane 4 is Escherichia coli O157:H7 E043,
  • lane 5 is Escherichia coli O157:H7 E044, NG Is blank control
  • Figure B is the color result of E.coli O157:H7 detected by cross-primer thermostatic amplification reaction technology (1 is Escherichia coli O157:H7 ATCC43895, 2 is Escherichia coli O157:H7 ATCC43
  • Figure 2 shows the gel electrophoresis results of the optimal amplification time test for detecting E. coli O157:H7 by cross isothermal amplification reaction; among them, lane 1 is the amplification time of 10 minutes, lane 2 is the amplification time of 20 minutes, and lane 3 The amplification time is 30 minutes, lane 4 is amplification time 40 minutes, lane 5 is amplification time 50 minutes, lane 6 is amplification time 60 minutes, lane 7 is amplification time 70 minutes, and lane 8 is amplification time 80 Minutes, NG is a blank control.
  • Figure 3 shows the experimental results of detecting the specificity of the target rfbE; among them, 1: E. coli O157: H7 ATCC43895, 2: E. coli O157: H7 ATCC 43894, 3: E. coli O157: H7 E019, 4: E.
  • Figure 4 is the result of the sensitivity experiment for detecting the target rfbE
  • Figure A is the result of the sensitivity test for detecting E. coli O157:H7 by the cross constant temperature amplification reaction
  • Figure B is the sensitivity test for detecting E. coli O157:H7 by the PCR amplification reaction
  • 1 is 3.28ng/ ⁇ L
  • 2 is 328pg/ ⁇ L
  • 3 is 32.8pg/ ⁇ L
  • 4 is 3.28pg/ ⁇ L
  • 5 is 328fg/ ⁇ L
  • 6 is 32.8fg/ ⁇ L
  • 7 is 3.28fg/ ⁇ L
  • NG is a negative control.
  • Figure 5 shows the gel electrophoresis results of the detection of E. coli Shiga toxin type I by the cross-primer isothermal amplification reaction technology;
  • Figure A shows the electrophoresis results of the first set of primers designed based on the target stx1, and
  • Figure B shows the results based on the target Electrophoresis results of the second set of primers designed by stx1 (lane 1 is E. coli O157:H7 ATCC43895, lane 2 is E. coli O157:H7 ATCC43894, lane 3 is E. coli O157:H7 E019, lane 4 is E. coli O157:H7 E043, lane 5 is E. coli O157:H7 E044, NG is blank control).
  • the method for detecting E. coli O157:H7 based on cross-primer isothermal amplification reaction technology includes the following steps:
  • a method for detecting pathogenic microorganisms based on the cross-primer isothermal amplification reaction technology E. coli O157:H7 is taken as an example, and the reagents used are as follows:
  • reaction stock solution composed of 40.0mM Tris-HCl, 20.0mM ammonium sulfate, 20.0mM potassium chloride, 16.0mM magnesium sulfate, 0.2%(v/v) Tween 20, 1.4M Betaine, 10.0mM dNTPs (each) composition;
  • Bst DNA polymerase large fragment, NEB company
  • an experimental group and a blank control group are set at the same time.
  • the experimental group is five strains of E.coli O157:H7, which are Escherichia coli O157:H7 ATCC43895, Escherichia coli O157:H7 ATCC43894, Escherichia coli O157:H7 E019, and Escherichia coli O157:H7 E043, Escherichia coli O157:H7 E044; all strains can be obtained from public sources;
  • the DNA extraction kit (Guangdong Dongsheng Biotechnology Co., Ltd.) was used to extract the bacterial DNA of each group and operate according to the kit instructions.
  • the OD 260 /OD 280 value of the bacterial DNA aqueous solution obtained in the experimental group (the ratio of absorbance at 260 nm and 280 nm) is 1.8.
  • the concentration of each substance is: Tris-HCl 20.0mM, ammonium sulfate 10.0mM, potassium chloride 10.0mM, magnesium sulfate 8.0mM, Tween 20 0.1% (v/v), betaine 0.7M, dNTPs(each) 1.4mM , Bst DNA polymerase 8U, stripping primer 4s, 5a each 0.6 ⁇ M, cross primer 2a1s 1.0 ⁇ M, specific primer 2a and 3a each 0.5 ⁇ M.
  • the reaction tube was kept in a water bath at 63°C for 60 minutes, and then kept in a water bath at 80°C for 2 minutes to terminate the reaction.
  • the results are shown in Figure 1.
  • the results show that the color of the blank control group is yellow, indicating that it does not contain E. coli O157:H7; the color of the experimental group changes to green, indicating that it contains E. coli O157:H7.
  • the amplified product was subjected to 2% agarose gel electrophoresis. The positive group showed a trapezoidal band, and the negative group had no amplified band, which was consistent with the expected result.
  • the optimum amplification time test for detecting E. coli O157:H7 by cross constant temperature amplification reaction includes the following steps:
  • the reaction system was constructed according to Example 1, sterile water was used as a blank control, and the amplified products were subjected to 2% agarose gel electrophoresis.
  • the results are shown in Figure 2.
  • the ladder-like bands appear when the reaction time is from 40 minutes to 90 minutes, while the ladder-like bands from 60 minutes to 90 minutes have no significant difference, so the reaction time for constant temperature amplification of cross primers can be reduced. It is 60 minutes, which can achieve the shortest amplification time and the best amplification effect.
  • the cross constant temperature amplification reaction to detect E. coli O157:H7 specificity test includes the following steps:
  • the genomic DNA of Escherichia coli O157:H7 ATCC43895 and non-E.coli genomic DNA was established according to the reaction system and conditions in Example 1 to establish a cross constant temperature amplification reaction detection method, and conduct a specific test;
  • the non-E.coli are: Salmonella ATCC29629; Salmonella ATCC19585; Salmonella ATCC14028; Salmonella ATCC13076; Listeria monocytogenes ATCC19116; Listeria monocytogenes ATCC19114; Listeria monocytogenes ATCC19115; Listeria monocytogenes ATCC15313; Listeria monocytogenes ATCC15313 Special bacteria ATCC19113; Pseudomonas aeruginosa ATCC27853; Staphylococcus aureus ATCC27664; Methicillin-resistant Staphylococcus aureus NCTC10442; Methicillin-resistant Staphylococcus aureus N315; Methicillin-resistant Staphylococcus aureus 85/2082; Tolerant Methicillin Staphylococcus aureus CA05; Vibrio parahaemolyticus ATCC17802; Vibrio parahaemolyticus ATCC27969; Lactobacillus casei BM
  • the comparison test of the sensitivity of cross constant temperature amplification reaction and PCR amplification reaction for detecting E. coli O157:H7 includes the following steps:
  • E. coli O157:H7 The genome of E. coli O157:H7 was diluted by 10-fold concentration, respectively, 3.28ng/ ⁇ L; 328pg/ ⁇ L; 32.8pg/ ⁇ L; 3.28pg/ ⁇ L; 328fg/ ⁇ L; 32.8fg/ ⁇ L; 3.28fg/ ⁇ L; 328ag/ ⁇ L, and set a negative control (de-nucleic acid water) at the same time, construct a cross constant temperature amplification method according to the reaction system in Example 1, and perform 2% agarose gel electrophoresis on the amplified products to determine the sensitivity of the detection method.
  • a negative control de-nucleic acid water
  • Figure A is the result of the sensitivity test for detecting E. coli O157:H7 by the cross isothermal amplification reaction
  • Figure B is the sensitivity test for detecting E. coli O157:H7 by the PCR amplification reaction. It can be seen from Figure A that all samples with a concentration of E. coli DNA higher than 3.28 fg/ ⁇ L have trapezoidal bands, showing a positive result. In Figure B, a single band appears when the concentration of E. coli DNA in the sample is higher than 32.8 pg/ ⁇ L.
  • the screening of primers for detection of E. coli Shiga toxin type I by cross isothermal amplification reaction includes the following steps:
  • PrimerPremier software design two sets of primers for the stx1 target.
  • the primer sequences are as follows (5’-3’):
  • Target stx1-1 stripping primer 4s 5’-agttgatgtcagagggatag-3’ (SEQ ID NO.6);
  • Target stx1-1 stripping primer 5a 5’-cgctgttgtacctggaaa-3’ (SEQ ID NO. 7);
  • Target stx1-1 cross primer 2a1s 5’-atcagcaaagcgataaaa ctacggcttattgttgaa-3’ (SEQ ID NO. 8);
  • Target stx1-1 specific primer 2a 5'-atcagcaaagcgataaaa-3' (SEQ ID NO. 9).
  • Target stx1-1 specific primer 3a 5’-cctgttaacaaatcctgtcac-3’ (SEQ ID NO.10)
  • Target stx1-2 stripping primer 4s 5’-gagcgatgttacggtttg-3’ (SEQ ID NO.11);
  • Target stx1-2 stripping primer 5a 5’-caggcaggacactactcaa-3’ (SEQ ID NO.12);
  • Target stx1-2 cross primer 2a1s 5’-caacatcttcagcagtcatggggatttcgtacaacac-3’ (SEQ ID NO.13);
  • Target stx1-2 specific primer 2a 5'-caacatcttcagcagtcat-3' (SEQ ID NO.14).
  • Target stx1-2 specific primer 3a 5'-gaacgcccactgagatcatcc-3' (SEQ ID NO. 15).
  • Escherichia coli O157:H7 ATCC43895, Escherichia coli O157:H7 E019, Escherichia coli O157:H7 E020, Escherichia coli O157:H7 E043, Escherichia coli O157:H7 E044 were established according to the reaction system and conditions in Example 1.
  • the amplified products were subjected to 2% agarose gel electrophoresis.
  • Figure A is the electrophoresis result of the first set of primers designed according to the target stx1
  • Figure B is the electrophoresis result of the second set of primers designed according to the target stx1.
  • the blank control group in the first set of primers designed by target stx1 has no obvious ladder-like strips, while the blank control group of the second set of primers has obvious ladder-like strips, indicating that the quality of the first set of primers is higher. It can effectively reduce the false positives in the constant temperature amplification reaction of cross primers.
  • the detection limit for E. coli O157:H7 rfbE is 3.28fg/ ⁇ L, which is about 10,000 times that of conventional PCR reactions, and has high sensitivity.

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Abstract

本发明公开了一种针对大肠杆菌O157:H7的特异性靶序列rfbE及stx1设计的CPA引物及其检测试剂盒和检测方法。

Description

大肠杆菌O157:H7的CPA引物及试剂盒和检测方法 技术领域
本发明属于生物技术领域，具体涉及一种大肠杆菌O157:H7的CPA引物及其检测试剂盒和检测方法。
背景技术
目前微生物的检测鉴定方法主要分为培养鉴定法、免疫检测法及核酸检测。培养鉴定法及免疫检测法操作繁琐、实验周期长，对实验人员专业水平要求高。
交叉引物恒温扩增(Crossing Priming Amplification,CPA)技术与其他核酸扩增技术相比，可以在等温条件下快速、高效、特异地扩增靶序列，且操作简便，不需要精准的变温设备，成本较低，在食源性微生物检测领域显示出广阔的发展前景。但该反应引物设计难度较高，需要在有限的产物长度中设计五条引物，且避免五条引物自身发生非特异性扩增而影响结果，因此引物的设计尤为重要。
Escherichia coli O157:H7是肠出血大肠杆菌最重要的血清型，是一种人畜共患的肠道致病菌，主要通过污染的动物源性食品感染人类，如生的或绞碎的肉制品、生牛奶和受污染的蔬菜和芽菜向人类传播，感染人体后表现为相应的临床症状。轻度感染可引起腹部绞痛和腹泻，发烧和呕吐也可能出现。重度感染则表现为出血性结肠炎，溶血性尿毒综合征。
目前由E.coli O157:H7感染引起的食源性疾病已经成为世界性的卫生问题。而在E.coli O157:H7中存在部分可以产志贺毒素I型的大肠杆菌。若发生感染可导致人类出现腹泻、出血性结肠炎等一些胃肠道疾病。志贺毒素I型在肠腔产生后，可穿过上皮细胞进入血循环，引起特定靶组织的血管损伤，如脑、肾血管损伤。产志贺毒素大肠杆菌具有强烈的致病性和致死性等特点，严重威胁人类的生命健康，已成为世界性的公共卫生问题。
中国专利申请CN 108796098A公开了一种PSR等温扩增反应检测大肠杆菌 志贺毒素的引物、试剂盒及其方法，该专利针对志贺毒素特异性靶序列stx2设计了PSR反应的引物用于检测。
中国专利申请CN 108796099A公开了一种大肠杆菌O157:H7的PSR检测引物、试剂盒及其检测方法，该专利针对大肠杆菌O157:H7的特异性靶序列rfbE设计了PSR反应的引物用于检测。
以上专利中的反应灵敏度最小单位为pg/μL，灵敏度不够高，限制了适用范围。
发明内容
为了克服PSR反应检测灵敏度低的问题，本发明的首要目的在于一种大肠杆菌O157:H7的CPA引物，其灵敏度达到了fg/μL，是PSR反应灵敏度的100～1000倍，能够解决现有技术灵敏度较低的问题。
本发明的另一目的在于提供一种大肠杆菌O157:H7的CPA检测试剂盒。
本发明的再一目的在于提供一种大肠杆菌O157:H7的CPA检测方法，该方法具有灵敏度高、特异性好，操作简便快速，结果准确可靠，检测成本低，适合现场检测应用的特点。
本发明的目的通过下述技术方案实现：
一种大肠杆菌O157:H7的CPA检测引物，是针对其靶点rfbE设计的，包括剥离引物4s、5a，交叉扩增引物2a1s，以及特异引物2a、3a；其核苷酸序列分别如下所示：
靶点rfbE剥离引物4s：5’-aggaccgcagaggaaaga-3’(SEQ ID NO.1)；
靶点rfbE剥离引物5a：5’-tccacgccaaccaagatc-3’(SEQ ID NO.2)；
靶点rfbE交叉引物2a1s：5’-agtacattggcatcgtgtcagataaactcatcgaaaca-3’(SEQ ID NO.3)；
靶点rfbE特异引物2a：5’-agtacattggcatcgtgt-3’(SEQ ID NO.4)；
靶点rfbE特异引物3a：5’-ggcatcgtgtggacagggt-3’(SEQ ID NO.5)。
一种大肠杆菌志贺毒素I型的CPA检测引物，是针对其靶点stx1设计的，包括剥离引物4s、5a，交叉扩增引物2a1s，以及特异引物2a、3a；其核苷酸序列分别如下所示：
靶点stx1剥离引物4s：5’-agttgatgtcagagggatag-3’(SEQ ID NO.6)；
靶点stx1剥离引物5a：5’-cgctgttgtacctggaaa-3’(SEQ ID NO.7)；
靶点stx1交叉引物2a1s：5’-atcagcaaagcgataaaactacggcttattgttgaa-3’(SEQ ID NO.8)；
靶点stx1特异引物2a：5’-atcagcaaagcgataaaa-3’(SEQ ID NO.9)；
靶点stx1特异引物3a：5’-cctgttaacaaatcctgtcac-3’(SEQ ID NO.10)。
一种大肠杆菌O157:H7的CPA检测试剂盒，包括上述的大肠杆菌O157:H7的CPA检测引物和/或大肠杆菌志贺毒素I型的CPA检测引物；
所述试剂盒中，各CPA检测引物的浓度优选10μM；
所述的试剂盒还包括如下组分：
A、2×反应缓冲液：40.0mM的Tris-HCl，20.0mM的硫酸铵，20.0mM的氯化钾，16.0mM的硫酸镁，0.2％(v/v)的Tween 20，1.4M的甜菜碱，10.0mM的dNTPs(each)；
B、Bst DNA聚合酶；
C、钙黄绿素和氯化锰的混合溶液；
组分B中所述的Bst DNA聚合酶优选浓度为8U/μL的Bst DNA聚合酶水溶液。
组分C中所述的钙黄绿素和氯化锰的混合溶液通过如下方法制备得到：
(i)将钙黄绿素溶于二甲基亚砜(DMSO)中，配制50μM的钙黄绿素溶液；将氯化锰溶于水中，配制1mM的氯化锰水溶液；
(ii)取25μL 50μM的钙黄绿素溶液与10μL1mM的氯化锰水溶液混合均匀，得到钙黄绿素和氯化锰的混合溶液(钙黄绿素溶液与氯化锰溶液的浓度比为1:8)。
一种大肠杆菌O157:H7的CPA检测方法，包括如下步骤：
(1)提取待检样品的细菌DNA作为模板DNA，并控制模板DNA水溶液的OD 260/OD 280值为1.8～2.0；
(2)分别建立检测rfbE和/或stx1的交叉引物恒温扩增反应体系，于63℃水浴中保温至少60分钟进行交叉引物恒温扩增反应，待反应完成后于80℃水浴中保温2分钟终止反应；
其中，交叉引物恒温扩增反应体系为：2×反应缓冲液12.5μL，10μM的剥离引物4s和10μM的剥离引物5a各1.5μL，10μM的交叉引物2a1s 2.5μL，10μM的特异引物2a和10μM的特异引物3a各1.25μL，DNA模板1.0μL，8U/μL的Bst DNA聚合酶1.0μL，加去核酸水补足至25μL；最后加入1μL的钙黄绿素与氯化锰的混合溶液；
(3)针对靶点rfbE的检测，终止反应后肉眼观察反应体系的颜色，如颜色为黄色，说明待检样品中不含有大肠杆菌O157:H7；如颜色变为绿色，说明待检样品中含有大肠杆菌O157:H7；
(4)针对靶点stx1的检测，终止反应后对扩增产物做琼脂糖凝胶电泳，呈现梯形条带的含有大肠杆菌志贺毒素I型，无扩增条带的不含有大肠杆菌志贺毒素I型。
本发明相对于现有技术具有如下的优点及效果：
(1)本发明中所提供的针对E.coliO157:H7特异性靶序列rfbE及stx1所设计的交叉引物恒温扩增反应检测鉴定体系，解决现有技术方法所需周期长，灵敏度低，成本高，现场应用困难等缺陷。通过选择目标菌株的特异性序列rfbE及stx1的保守区域，设计一对剥离引物、交叉引物和特异引物构建交叉引物恒温扩增反应体系，并在60分钟左右获得检测结果以缩短传统大肠杆菌检测的周期。
(2)目前，有文献报道采用交叉引物恒温扩增反应技术检测大肠杆菌O157:H7,但是检测灵敏度不高，相较于普通PCR没有体现交叉引物恒温扩增技术灵敏度方面显著的优越性。而本发明可以降低出现假阳性结果的概率，同时确保检测的可靠性、特异性和高灵敏度。这对于提高重大群体疾病诊断率，早期的疾病诊断具有非常重要的意义。
(3)本发明在恒温条件下扩增，不会因温度的改变而造成时间损失，耗时短，此外，该技术不需要特殊、昂贵的仪器和试剂，扩增产物不需要凝胶电泳，直接用荧光染料显色可凭肉眼判断结果，操作简便快捷，检测成本较低。本发明的试剂盒及方法特别适用中小型单位及现场检测。
附图说明
图1为交叉引物恒温扩增反应技术检测E.coliO157:H7的凝胶电泳结果及显色结果图；其中，图A为交叉引物恒温扩增检测E.coliO157:H7的凝胶电泳结果(泳道1为大肠杆菌O157:H7 ATCC43895，泳道2为大肠杆菌O157:H7 ATCC43894，泳道3为大肠杆菌O157:H7 E019，泳道4为大肠杆菌O157:H7 E043，泳道5为大肠杆菌O157:H7 E044，NG为空白对照)；图B为交叉引物恒温扩增反应技术检测E.coliO157:H7的显色结果(1为大肠杆菌O157:H7 ATCC43895，2为大肠杆菌O157:H7 ATCC43894，3为大肠杆菌O157:H7 E019，4为大肠杆菌O157:H7 E043，5为大肠杆菌O157:H7 E044，NG为空白对照)。
图2为交叉恒温扩增反应检测大肠杆菌O157:H7的最适扩增时间试验的凝胶电泳结果图；其中，泳道1为扩增时间10分钟，泳道2为扩增时间20分钟，泳道3为扩增时间30分钟，泳道4为扩增时间40分钟，泳道5为扩增时间50分钟，泳道6为扩增时间60分钟，泳道7为扩增时间70分钟，泳道8为扩增时间80分钟，NG为空白对照。
图3为检测靶点rfbE特异性的实验结果图；其中，1：大肠杆菌O157:H7 ATCC43895，2：大肠杆菌O157:H7 ATCC43894，3：大肠杆菌O157:H7 E019，4：大肠杆菌O157:H7 E043；5：大肠杆菌O157:H7 E044；6：沙门氏菌ATCC29629；7：沙门氏菌ATCC19585；8：沙门氏菌ATCC14028；9：沙门氏菌ATCC13076；10：单增李斯特菌ATCC19116；11：单增李斯特菌ATCC19114，12：单增李斯特菌ATCC19115；13：单增李斯特菌ATCC15313；14：单增李斯特菌ATCC19113；15：铜绿假单胞菌ATCC27853；16：金黄色葡萄球菌ATCC27664；17：耐甲氧西林金黄色葡萄球菌NCTC10442；18：耐甲氧西林金黄色葡萄球菌N315；19：耐甲氧西林金黄色葡萄球菌85/2082；20：耐甲氧西林金黄色葡萄球菌CA05；21：副溶血性弧菌ATCC17802；22：副溶血性弧菌ATCC27969；23：干酪乳杆菌BM-LC14617；24：阴性对照。
图4为检测靶点rfbE灵敏度实验结果图，图A为交叉恒温扩增反应检测大肠杆菌O157:H7的灵敏度试验的结果图，图B为PCR扩增反应检测大肠杆菌O157:H7的灵敏度试验；其中，1为3.28ng/μL；2为328pg/μL；3为32.8pg/μL；4为3.28pg/μL；5为328fg/μL；6为32.8fg/μL；7为3.28fg/μL；8为328ag/μL；NG为阴性对照。
图5为交叉引物恒温扩增反应技术检测大肠杆菌志贺毒素I型的凝胶电泳结果；其中，图A为根据靶点stx1所设计的第一套引物的电泳结果，图B为根据靶点stx1所设计的第二套引物的电泳结果(泳道1为大肠杆菌O157:H7 ATCC43895，泳道2为大肠杆菌O157:H7 ATCC43894，泳道3为大肠杆菌O157:H7 E019,泳道4为大肠杆菌O157:H7 E043,泳道5为大肠杆菌O157:H7 E044，NG为空白对照)。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例1
基于交叉引物恒温扩增反应技术检测大肠杆菌O157:H7的方法，包括以下步骤：
1、基于交叉引物恒温扩增反应技术检测病原微生物的方法，本实施例以大肠杆菌O157:H7为例，其使用试剂如下：
a.浓度各为10μM的剥离引物4s、5a，交叉扩增引物2a1s，以及特异引物2a、3a，引物序列如前文SEQ ID NO.1-SEQ ID NO.5所示；
b.2×反应储液：由浓度为40.0mM的Tris-HCl，20.0mM的硫酸铵，20.0mM的氯化钾，16.0mM的硫酸镁，0.2％(v/v)的Tween 20，1.4M的甜菜碱，10.0mM的dNTPs(each)组成；
c.浓度为8U/μL的Bst DNA聚合酶(大片段，NEB公司)水溶液；
d.钙黄绿素和氯化锰的混合溶液：先配制浓度为50μM的钙黄绿素溶液(二甲基亚砜溶解)；然后取25μL 50μM的钙黄绿素溶液，与10μL1mM的氯化锰水溶液混合均匀(钙黄绿素溶液与氯化锰溶液的浓度比为1:8)。
2、使用上述试剂利用交叉引物恒温扩增反应技术检测大肠杆菌O157:H7，包括如下步骤：
(1)提取待检样品的细菌DNA作为模板DNA：
本实施例同时设置实验组和空白对照组，其中实验组为五株E.coli O157:H7，分别是大肠杆菌O157:H7 ATCC43895，大肠杆菌O157:H7 ATCC43894， 大肠杆菌O157:H7 E019,大肠杆菌O157:H7 E043,大肠杆菌O157:H7 E044；所有菌株均可由公开途径获得；
采用DNA提取试剂盒(广东东盛生物科技有限公司)提取各组细菌DNA，按照试剂盒说明书操作，实验组所得细菌DNA水溶液的OD 260/OD 280的值(260nm和280nm下吸光光度比值)为1.8。
(2)建立检测rfbE的交叉引物恒温扩增反应：
在反应管中配制总体积为26μL的交叉引物恒温扩增反应体系：加入2×反应储液12.5μL，4s与5a等体积混合引物混合液3.0μL，交叉引物2a1s 2.5μL，特异引物2a与3a等体积混合液2.5μL，Bst DNA聚合酶1μL，DNA模板1.0μL，用去核酸水补充体积至25μL，最后加入上述浓度的钙黄绿素及氯化锰混合液水溶液1μL，混匀即可。此时各物质浓度为：Tris-HCl 20.0mM，硫酸铵10.0mM，氯化钾10.0mM，硫酸镁8.0mM，Tween 20 0.1％(v/v)，甜菜碱0.7M，dNTPs(each)1.4mM，Bst DNA聚合酶8U，剥离引物4s、5a各0.6μM，交叉引物2a1s 1.0μM，特异引物2a及3a各0.5μM。将反应管置于63℃水浴中保温反应60分钟，再于80℃水浴中保温2分钟终止反应。
(3)显色检测：
待反应结束后，用肉眼观察颜色变化。
结果如图1所示，结果显示：空白对照组的颜色为黄色，说明不含有E.coli O157:H7菌；实验组的颜色变为绿色，说明含有E.coli O157:H7菌，随后对扩增产物进行2％的琼脂糖凝胶电泳，阳性组呈现梯形条带，阴性组无扩增条带，与预期结果一致。
实施例2
交叉恒温扩增反应检测大肠杆菌O157:H7的最适扩增时间试验，包括以下步骤：
按照实施例1反应体系构建交叉恒温扩增方法，分别进行10分钟，20分钟，30分钟，40分钟，50分钟，60分钟，70分钟，80分钟，90分钟的不同扩增时间试验，以确定最适的扩增时间。
按照实施例1构建反应体系，无菌水作空白对照，并对扩增产物进行2％的琼 脂糖凝胶电泳。
结果如图2所示，反应时间为40分钟至90分钟时均出现了梯状条带，而60分钟至90分钟的梯状条带无显著差别，所以可将交叉引物恒温扩增反应时间减少为60分钟，能够实现最短的扩增时间和最佳的扩增效果。
实施例3
交叉恒温扩增反应检测大肠杆菌O157:H7特异性试验，包括以下步骤：
将大肠杆菌O157:H7 ATCC43895与非大肠杆菌的基因组DNA按照实施例1中的反应体系和条件建立交叉恒温扩增反应检测方法，进行特异性试验；
其中，非大肠杆菌为：沙门氏菌ATCC29629；沙门氏菌ATCC19585；沙门氏菌ATCC14028；沙门氏菌ATCC13076；单增李斯特菌ATCC19116；单增李斯特菌ATCC19114；单增李斯特菌ATCC19115；单增李斯特菌ATCC15313；单增李斯特菌ATCC19113；铜绿假单胞菌ATCC27853；金黄色葡萄球菌ATCC27664；耐甲氧西林金黄色葡萄球菌NCTC10442；耐甲氧西林金黄色葡萄球菌N315；耐甲氧西林金黄色葡萄球菌85/2082；耐甲氧西林金黄色葡萄球菌CA05；副溶血性弧菌ATCC17802；副溶血性弧菌ATCC27969；干酪乳杆菌BM-LC14617。
设置大肠杆菌O157:H7基因组为阳性对照，去核酸水为阴性对照。对扩增产物进行2％的琼脂糖凝胶电泳，结果如图3所示，加入大肠杆菌基因组的反应体系呈现梯形条带，阴性组无扩增条带，与预期结果一致。
如此表明，基于交叉引物恒温扩增反应检测E.coliO157:H7的引物具有较高的特异性。
实施例4
交叉恒温扩增反应和PCR扩增反应检测大肠杆菌O157:H7的灵敏度对比试验，包括以下步骤：
1.交叉恒温扩增反应检测大肠杆菌O157:H7的灵敏度试验
将大肠杆菌O157:H7的基因组进行10倍浓度梯度稀释，分别为3.28ng/μL；328pg/μL；32.8pg/μL；3.28pg/μL；328fg/μL；32.8fg/μL；3.28fg/μL；328ag/μL，同时设置阴性对照(去核酸水)，按照实施例1中的反应体系构建交叉恒温扩增 方法并对扩增产物进行2％的琼脂糖凝胶电泳，以确定检测方法的灵敏度。
2.PCR扩增反应检测大肠杆菌O157:H7的灵敏度试验
(1)基于PCR扩增反应技术检测病原微生物的方法，本实施例以大肠杆菌O157:H7为例，其使用试剂如下：
a.浓度各为10μM的剥离引物4s、5a，引物序列如前文SEQ ID NO.1、SEQ ID NO.2所示；
b.2×Taq PCR MasterMix；
(2)建立检测rfbE的PCR扩增反应：
在反应管中配制总体积为25μL的交叉引物恒温扩增反应体系：加入2×Taq PCR MasterMix 12.5μL，4s与5a等体积混合引物混合液3.0μL，DNA模板2.0μL，用去核酸水补充体积至25μL。将反应管置于PCR仪中进行扩增。
(3)将大肠杆菌O157:H7的基因组进行10倍浓度梯度稀释，分别为3.28ng/μL；328pg/μL；32.8pg/μL；3.28pg/μL；328fg/μL；32.8fg/μL；3.28fg/μL；328ag/μL，同时设置阴性对照(去核酸水)。按照实施例4中的PCR扩增反应体系构建PCR扩增方法并对扩增产物进行2％的琼脂糖凝胶电泳，以确定检测方法的灵敏度。阳性组呈现单一条带，阴性组无扩增条带，与预期结果一致。
结果如图4所示，图A为交叉恒温扩增反应检测大肠杆菌O157:H7的灵敏度试验的结果图，图B为PCR扩增反应检测大肠杆菌O157:H7的灵敏度试验。从图A中可以看出，样品中大肠杆菌DNA的浓度高于3.28fg/μL的均出现了梯形条带，呈现阳性结果。而图B中，当样品中大肠杆菌DNA的浓度高于32.8pg/μL时才出现了单一条带。
结果表明：建立的E.coli O157:H7交叉恒温扩增反应方法可检测样品中3.28fg/μL反应的大肠杆菌DNA，其灵敏度是PCR扩增反应检测的10000倍。
实施例5
交叉恒温扩增反应检测大肠杆菌志贺毒素I型的引物筛选，包括以下步骤：
1.根据CPA扩增反应原理，使用PrimerPremier软件针对stx1靶点分别设计的两组引物，引物序列如下(5’-3’)：
靶点stx1-1剥离引物4s：5’-agttgatgtcagagggatag-3’(SEQ ID NO.6)；
靶点stx1-1剥离引物5a：5’-cgctgttgtacctggaaa-3’(SEQ ID NO.7)；
靶点stx1-1交叉引物2a1s：5’-atcagcaaagcgataaaa ctacggcttattgttgaa-3’(SEQ ID NO.8)；
靶点stx1-1特异引物2a：5’-atcagcaaagcgataaaa-3’(SEQ ID NO.9)。
靶点stx1-1特异引物3a：5’-cctgttaacaaatcctgtcac-3’(SEQ ID NO.10)
靶点stx1-2剥离引物4s：5’-gagcgatgttacggtttg-3’(SEQ ID NO.11)；
靶点stx1-2剥离引物5a：5’-caggcaggacactactcaa-3’(SEQ ID NO.12)；
靶点stx1-2交叉引物2a1s：5’-caacatcttcagcagtcatggggatttcgtacaacac-3’(SEQ ID NO.13)；
靶点stx1-2特异引物2a：5’-caacatcttcagcagtcat-3’(SEQ ID NO.14)。
靶点stx1-2特异引物3a：5’-gaacgcccactgagatcatcc-3’(SEQ ID NO.15)。
2、将大肠杆菌O157:H7 ATCC43895，大肠杆菌O157:H7 E019，大肠杆菌O157:H7 E020，大肠杆菌O157:H7 E043，大肠杆菌O157:H7 E044的基因组按照实施例1中的反应体系和条件建立交叉恒温扩增反应检测方法，进行大肠杆菌志贺毒素I型的检测，去核酸水为阴性对照。对扩增产物进行2％的琼脂糖凝胶电泳。
结果如图5所示，图A为根据靶点stx1所设计的第一套引物的电泳结果，图B为根据靶点stx1所设计的第二套引物的电泳结果。靶点stx1所设计的第一套引物中的空白对照组无明显梯状条带，而第二套引物的空白对照组出现了明显的梯状条带，说明第一套引物的质量更高，能够有效减少交叉引物恒温扩增反应中假阳性的产生。
结论：从上述实验结果可以看出，交叉恒温扩增反应扩增方法与常规PCR和荧光PCR具有如下优点：
操作和鉴定简便快捷：常规PCR整个过程在2～4个小时才能出结果，荧光定量PCR需要2～3小时，本发明所提供的检测方法在60分钟就可出现阳性结果。其次对仪器要求低，仅需要一个普通水浴锅，并可以通过荧光染料直接观测检测结果，省去了传统的电泳检测步骤。在快速检测及现场检测的实践中有广泛的应用前景。
特异性强：仅通过是否扩增就可判断目的基因的存在与否，从而完成了细 菌的定性检测。
灵敏度高:针对大肠杆菌O157:H7 rfbE的检测限为3.28fg/μL，是常规PCR反应10000倍左右，具有较高的灵敏度。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Figure PCTCN2020074822-appb-000001
Figure PCTCN2020074822-appb-000002
Figure PCTCN2020074822-appb-000003
Figure PCTCN2020074822-appb-000004
Figure PCTCN2020074822-appb-000005

Claims (9)

  1. 一种大肠杆菌O157:H7的CPA检测引物，其特征在于：是针对靶点rfbE设计的，包括剥离引物4s、5a，交叉扩增引物2a1s，以及特异引物2a、3a；其核苷酸序列分别如下所示：
    靶点rfbE剥离引物4s：5’-aggaccgcagaggaaaga-3’(SEQ ID NO.1)；
    靶点rfbE剥离引物5a：5’-tccacgccaaccaagatc-3’(SEQ ID NO.2)；
    靶点rfbE交叉引物2a1s：5’-agtacattggcatcgtgtcagataaactcatcgaaaca-3’(SEQ ID NO.3)；
    靶点rfbE特异引物2a：5’-agtacattggcatcgtgt-3’(SEQ ID NO.4)；
    靶点rfbE特异引物3a：5’-ggcatcgtgtggacagggt-3’(SEQ ID NO.5)。
  2. 一种大肠杆菌志贺毒素I型的CPA检测引物，其特征在于：是针对靶点stx1设计的，包括剥离引物4s、5a，交叉扩增引物2a1s，以及特异引物2a、3a；其核苷酸序列分别如下所示：
    靶点stx1剥离引物4s：5’-agttgatgtcagagggatag-3’(SEQ ID NO.6)；
    靶点stx1剥离引物5a：5’-cgctgttgtacctggaaa-3’(SEQ ID NO.7)；
    靶点stx1交叉引物2a1s：5’-atcagcaaagcgataaaactacggcttattgttgaa-3’(SEQ ID NO.8)；
    靶点stx1特异引物2a：5’-atcagcaaagcgataaaa-3’(SEQ ID NO.9)；
    靶点stx1特异引物3a：5’-cctgttaacaaatcctgtcac-3’(SEQ ID NO.10)。
  3. 一种大肠杆菌O157:H7的CPA检测试剂盒，其特征在于包括权利要求1所述的CPA检测引物和/或权利要求2所述的CPA检测引物。
  4. 根据权利要求3所述的试剂盒，其特征在于：各CPA检测引物的浓度为10μM。
  5. 根据权利要求3所述的试剂盒，其特征在于还包括如下组分：
    A、2×反应缓冲液：40.0 mM的Tris-HCl，20.0mM的硫酸铵，20.0mM的氯化钾，16.0mM的硫酸镁，0.2％(v/v)的Tween 20，1.4 M的甜菜碱，10.0 mM的dNTPs；
    B、Bst DNA聚合酶；
    C、钙黄绿素和氯化锰的混合溶液。
  6. 根据权利要求5所述的试剂盒，其特征在于：组分B中所述的Bst DNA聚合酶是浓度为8U/μL的Bst DNA聚合酶水溶液。
  7. 根据权利要求5所述的试剂盒，其特征在于：组分C中所述的钙黄绿素和氯化锰的混合溶液通过如下方法制备得到：
    (i)将钙黄绿素溶于二甲基亚砜中，配制50μM的钙黄绿素溶液；将氯化锰溶于水中，配制1 mM的氯化锰水溶液；
    (ii)取25μL 50μM的钙黄绿素溶液与10μL1 mM的氯化锰水溶液混合均匀，得到钙黄绿素和氯化锰的混合溶液。
  8. 一种大肠杆菌O157:H7的CPA检测方法，其特征在于包括如下步骤：
    (1)提取待检样品的细菌DNA作为模板DNA，并控制模板DNA水溶液的OD 260/OD 280值为1.8～2.0；
    (2)分别建立检测rfbE和/或stx1的交叉引物恒温扩增反应体系，于63℃水浴中保温至少60分钟进行交叉引物恒温扩增反应，待反应完成后于80℃水浴中保温2分钟终止反应；
    (3)针对靶点rfbE的检测，终止反应后肉眼观察反应体系的颜色，如颜色为黄色，说明待检样品中不含有大肠杆菌O157:H7；如颜色变为绿色，说明待检样品中含有大肠杆菌O157:H7；
    (4)针对靶点stx1的检测，终止反应后对扩增产物做琼脂糖凝胶电泳，呈现梯形条带的含有大肠杆菌志贺毒素I型，无扩增条带的不含有大肠杆菌志贺毒素I型。
  9. 根据权利要求8所述的检测方法，其特征在于：步骤(2)所述的交叉引物恒温扩增反应体系为：2×反应缓冲液12.5μL，10μM的剥离引物4s和10μM的剥离引物5a各1.5μL，10μM的交叉引物2a1s 2.5μL，10μM的特异引物2a和10μM的特异引物3a各1.25μL，DNA模板1.0μL，8U/μL的Bst DNA聚合酶1.0μL，加去核酸水补足至25μL；最后加入1μL的钙黄绿素与氯化锰的混合溶液。
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