WO2020133638A1 - 一种检测mcr基因的试剂盒、检测方法及其应用 - Google Patents

一种检测mcr基因的试剂盒、检测方法及其应用 Download PDF

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WO2020133638A1
WO2020133638A1 PCT/CN2019/074897 CN2019074897W WO2020133638A1 WO 2020133638 A1 WO2020133638 A1 WO 2020133638A1 CN 2019074897 W CN2019074897 W CN 2019074897W WO 2020133638 A1 WO2020133638 A1 WO 2020133638A1
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seq
pcr
primer shown
mcr
primer
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胡双芳
柯跃斌
刘蔚雯
吕子全
沈建忠
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深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所）
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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    • C12Q1/686—Polymerase chain reaction [PCR]
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the invention belongs to the fields of medical hygiene and microbial detection, and a kit, a detection method and an application thereof for detecting any one of mcr-1 to mcr-6 in a stool sample.
  • Polymyxin is an antimicrobial peptide found in the culture medium of Paenibacillus polymyxa (Paenibacillus polymyxa).
  • Paenibacillus polymyxa There are five types, including A, B, C, D, and E, among which polymyxin E (colistin) and Polymyxin B (PMB) is the two most commonly used polymyxin in the clinic. Due to its neurotoxicity, its clinical use is strictly restricted, but in Asia, Europe and North America, polymyxin is widely used in the livestock industry. According to China's CHINET bacterial resistance monitoring, due to the high infection rate of Carbapenem-resistant Enterobacteriaceae (CRE), polymyxin is regarded as the last line of defense. In the treatment of critically ill patients, polymyxin is the most important antibacterial drug in rescue therapy strategies.
  • CRE Carbapenem-resistant Enterobacteriaceae
  • E.coli containing mcr-2 (LT598652) has been isolated from samples of animal origin in Belgium; it has been isolated from pork samples from China E.coli containing mcr-3 (KY924928); E.coli containing mcr-2 (LT598652) in pork samples from China; and mcr-4 (MF543359) in pork samples from European sources ) And mcr-5 (KY807921) of E. coli and Salmonella enterericaserovar Typhimurium; Moraxella containing mcr-6 (MF176240) was isolated from pork samples from the United Kingdom.
  • the name of the invention is multiplex PCR primers, kits and detection methods for detecting polymyxin resistance genes, which are passed in mcr-1, mcr-2, mcr-3, mcr-4, mcr-5 Primer design based on the gene sequence, using different sizes of PCR products to distinguish the corresponding drug resistance genes, after optimizing the reaction system and conditions, a set of more complete multiple PCR reaction system was finally determined, which can be related to MCR Resistance genes are tested more comprehensively.
  • the invention patent application has the following defects: (1) the common PCR followed by gel electrophoresis method is likely to cause aerosol contamination and affect the detection results; (2) all its multiple primers are added to a reaction system, It is difficult to avoid the mutual reaction between primers, so non-specific amplification occurs, which affects the detection efficiency and detection quality of multiple PCR; (3) The real-time monitoring of PCR products is impossible, and it is not suitable for modern clinical detection and drug resistance monitoring and Requirements for rapid, sensitive and high-throughput screening.
  • the primary objective of the present invention is to provide a kit for detecting mobile polymyxin resistance MCR gene, which is rarely used in the prior art for molecular detection covering all MCR genes (mcr-1 to mcr-6) Method, this kit is based on the discovered MCR gene sequences, designed for all MCR detection fluorescent PCR detection method, this kit is particularly suitable for medical clinical or environmental monitoring scenarios, can quickly and easily and effectively in stool samples Detection and early warning of any MCR gene, real-time monitoring of PCR products, will greatly reduce the labor and material consumption of monitoring mobile polymyxin resistance genes, simplify the daily production and sampling of large numbers of clinical samples monitor.
  • the PCR-A reaction system of this kit uses a combination of a large number of universal primer pairs and a very small number of specific primers.
  • the specific primers specifically bind to the target fragment and are amplified.
  • the amplified fragment is amplified by the template. This combination can minimize the mutual reaction between primers and improve the efficiency of multiple PCR.
  • This kit uses fluorescent PCR technology as the detection method. The entire reaction is performed in a closed reaction tube. To avoid false positive results caused by PCR-electrophoresis and other aerosol contamination.
  • Another object of the present invention is to provide a detection method of the kit.
  • Another object of the present invention is to provide the application of a kit for detecting a mobile polymyxin resistance MCR gene in clinical or environmental monitoring.
  • a kit for detecting movable polymyxin resistance MCR gene which includes PCR-A group primers and PCR-B group primers
  • the PCR-A group primers include: SEQ ID No: 1 and SEQ ID No: 2 primer pairs, SEQ ID No: 3 and SEQ ID No: 4 primer pairs, SEQ ID No: 5 and SEQ ID No: 6 primer pairs, SEQ ID No: 7 and SEQ
  • the PCR-B group primers include: SEQ ID No: 11 and SEQ ID No: 12 Primer pairs, primer pairs shown in SEQ ID No: 13 and SEQ ID No: 14
  • the kit further includes: 2 ⁇ PCR buffer, Taq enzyme, dNTP, MgCl 2 , fluorescent dye EvaGreen and FTA test paper, 10% SDS solution, TE buffer.
  • the kit is a kit of 20-50 ⁇ L fluorescent PCR reaction system, each component and its content are as follows:
  • a detection method of a kit for detecting a movable polymyxin resistance MCR gene includes the following steps:
  • step (3) Add the DNA extracted in step (1) as a template to the PCR-A reaction system and the PCR-B reaction system respectively to perform PCR amplification reaction and perform fluorescent PCR detection; when the PCR-A reaction system and PCR-B When the actual melting curve Tm value of any system in the reaction system matches the standard melting curve Tm value of any kind of MCR gene, the sample to be tested has the MCR gene.
  • step (1) specifically includes the following steps: take 20 ⁇ L of the sample to be tested, place it in a centrifuge tube with a FTA filter membrane with a diameter of 2.0 mm, and then dry at 56° C. The dried FTA filter membrane is added with 10% SDS The solution was 200 ⁇ L, boiled for 10 min, washed twice with FTA special buffer, and then washed twice with TE buffer. After drying at 56°C, it could be used as a PCR reaction template.
  • the kit further includes: 2 ⁇ PCR buffer, Taq enzyme, dNTP, MgCl 2 , fluorescent dye EvaGreen and FTA test paper, 10% SDS solution, TE buffer.
  • the kit is a kit of 20-50 ⁇ L fluorescent PCR reaction system, each component and its content are as follows:
  • dNTPs 0.15mmol/L 0.15mmol/L Taq enzyme 2U 2U 20 ⁇ EvaGreen fluorescent dye 1 ⁇ 1 ⁇ Primer shown in SEQ ID No: 1 0.8 ⁇ mol/L no Primer shown in SEQ ID No: 2 0.8 ⁇ mol/L no Primer shown in SEQ ID No: 3 0.05 ⁇ mol/L no Primer shown in SEQ ID No: 4 0.05 ⁇ mol/L no Primer shown in SEQ ID No: 5 0.05 ⁇ mol/L no Primer shown in SEQ ID No: 6 0.05 ⁇ mol/L no Primer shown in SEQ ID No: 7 0.05 ⁇ mol/L no Primer shown in SEQ ID No: 8 0.05 ⁇ mol/L no Primer shown in SEQ ID No: 9 0.05 ⁇ mol/L no Primer shown in SEQ ID No: 10 0.05 ⁇ mol/L no Primer shown in SEQ ID No: 11 no 0.5 ⁇ mol/L Primer shown in SEQ ID No: 12 no 0.5 ⁇ mol/L Primer shown in SEQ ID No:
  • a kit for detecting movable polymyxin resistance MCR gene is used in clinical or environmental monitoring to detect MCR.
  • the present invention has the following advantages and effects:
  • this kit is designed to be suitable for all MCR detection based on the gene sequence of all MCRs found PCR detection method.
  • This kit is especially suitable for medical clinical or environmental monitoring scenarios, which can quickly and easily and effectively detect and warn any MCR gene in stool samples, and monitor PCR products in real time, which will greatly reduce the monitoring mobility
  • the labor and material consumption of polymyxin resistance genes simplifies the sampling and monitoring of large numbers of clinical samples in daily production and life. Therefore, this kit and its detection method have great application value in medical clinical and environmental monitoring scenarios .
  • the conventional method of Shen, et al 2018 (mentioned in Example 2) monitors mcr-1 and mcr-3 in the environment and the population.
  • the process includes selective screening bacteria, mass spectrometry to identify strains, drug sensitivity detection and PCR verification requires a total duration of 5 to 7 days; this method can reduce the selective screening step and directly detect the target gene from the total environmental DNA, the time can be shortened to 6 to 24h.
  • the PCR-A reaction system of this kit uses a combination of a large number of universal primer pairs and a very small number of specific primers.
  • the specific primers specifically bind to the target fragment and are amplified.
  • the amplified fragments are amplified by the template. Such a combination can minimize the mutual reaction between primers and improve the efficiency of multiplex PCR.
  • This kit uses fluorescent PCR technology as the detection method. The entire reaction is performed in a closed reaction tube, which avoids false positive results caused by PCR-electrophoresis and other aerosol contaminations.
  • the primers provided by the present invention have no amplification signal for the detection samples that do not contain MCR, indicating that they have good specificity.
  • Figure 1 is a SeqMan sequence analysis diagram of 46 MCR genes in the NCBI database.
  • Figures 3a-A and 3a-B are the dissolution curves of samples with a mixture of mcr-1 and mcr-3, where 3a-A is the dissolution curve obtained by the PCR-A reaction system, and 3a-B is the one obtained by the PCR-B reaction system Dissolution curve
  • Figures 3b-A and 3b-B are the dissolution curves of samples with a mixture of mcr-2, mcr-5 and mcr-6, where 3b-A is the dissolution curve obtained by the PCR-A reaction system, and 3b-B is PCR-B The dissolution curve obtained by the reaction system;
  • Figures 3c-A and 3c-B are the dissolution curves of samples with a mixture of mcr-3 and mcr-4, where 3c-A is the dissolution curve obtained by the PCR-A reaction system and 3c-B is the one obtained by the PCR-B reaction system Dissolution curve.
  • the ordinate of the dissolution curve is the Derivative derivative, specifically the change of the fluorescence intensity to the change of temperature, that is, d(Fluorescence)/d(T) or d(fluorescence value)/d(temperature); the abscissa is Temperature Temperature (°C), that is, each temperature point in the dissolution process.
  • the peak is the dissolution temperature Tm.
  • Primer sequence design in the kit of the present invention by analyzing all known MCR gene sequences separately, mcr-1 to mcr-5 are located in plasmid or group genes, mcr-6 is located in group genes, a total of 46 Sequence analysis of genes using SeqMan ( Figure 1), through analysis, to find highly conserved segments, through Premier 3.0 to select non-secondary structure and highly conserved segments, design multiple sets of primers, primer length is generally 20 bases There is no complementary sequence between primers and within primers. According to the pre-experimental results, there is a cross-reaction between the primer pair of mcr-1 and the primer of mcr-3, which can be used as a template to amplify each other. The T m value of the amplified fragment is about 79.9°C, so the pair is to avoid the reaction between the primers. , Divide the primers into two groups, A and B, primer list 1 is as follows:
  • the target area template used in the establishment and optimization of the reaction system is obtained as follows: the Escherichia coli and Klebsiella pneumoniae strains carrying mcr-1 to mcr-6, respectively, are recovered After 48 hours of incubation, take 1 mL of the culture solution, wash it with sterile water, and reconstitute it to 1 mL. Use phenol-chloroform method or kit to extract genomic nucleic acid, and then use the above primers for PCR amplification, and take the Ct value of 24-27 The latter is used as a template for the optimization of the reaction system in the future.
  • the initial reaction system (40 ⁇ L) is shown in Table 2:
  • reaction A The concentration of universal primers (primers shown in SEQ ID No: 1 and SEQ ID No: 2) is 0.8 ⁇ mol/L, and the concentration of specific primers (primers shown in SEQ ID No: 3 to SEQ ID No: 10) is 0.05 ⁇ mol/L L;
  • reaction B the concentration of specific primers (primers shown in SEQ ID No: 11 to SEQ ID No: 14) is 0.5 ⁇ mol/L.
  • Taq enzyme 2.3 Optimization of the amount of Taq DNA polymerase (Taq enzyme): under the premise that the final concentration of the primer is 0.2 ⁇ mol/L, the final concentration of magnesium ion is 2.5 mmol/L and other conditions in the reaction system are the same as Table 2, by comparing Taq The optimization experiment result of enzyme dosage (in units) was selected as 2U as the dosage of Taq enzyme in the kit reaction system.
  • dNTPs deoxyribonucleoside triphosphate
  • the unsaturated dye SYBRGREEN Due to the large number of primers and target fragments involved in this method, the unsaturated dye SYBRGREEN has a large error and cannot distinguish single gene differences. Therefore, this method uses the saturated dye EvaGreen as the fluorescent dye in the PCR process. Evagreen has minimal interference with qPCR, there is no "dye redistribution", saturation is embedded in DNA double strands, and the resolution effect is much better than SYBR Green.
  • the final determination of the fluorescent PCR reaction system is 40 ⁇ L system, the required components and corresponding concentrations are shown in Table 3.
  • each reagent should be adjusted in proportion.
  • the PCR-A reaction system is: every 40 ⁇ L PCR system includes 20 ⁇ L of 2 ⁇ PCR buffer; 0.4 ⁇ L of Taq enzyme at a concentration of 5 U/ ⁇ L; 4 ⁇ L of dNTP at a concentration of 10 mmol/L; 3.2 ⁇ L of MgCl 2 at a concentration of 25 mmol/L;
  • the primer shown in SEQ ID No: 1 and the primer shown in SEQ ID No: 2 were each 3.2 ⁇ L at a concentration of 10 ⁇ mol/L, the primer shown in SEQ ID No: 3 at 10 ⁇ mol/L, the primer shown in SEQ ID No: 4, and SEQ
  • the PCR-B reaction system is: every 40 ⁇ L PCR system includes 20 ⁇ L of 2 ⁇ PCR buffer; 0.4 ⁇ L of Taq enzyme at a concentration of 5 U/ ⁇ L; 4 ⁇ L of dNTP at a concentration of 10 mmol/L; 3.2 ⁇ L of MgCl 2 at a concentration of 25 mmol/L; 2 ⁇ L each of primers shown in SEQ ID No: 11, SEQ ID No: 12, SEQ ID No: 13 and SEQ ID No: 14 at a concentration of 10 ⁇ mol/L, 2 ⁇ L of template DNA; fluorescent dye 20 ⁇ EvaGreen 2 ⁇ L; make up to 40 ⁇ L with nucleic acid-free water.
  • the detection method of the kit of the present invention specifically includes the following steps:
  • the DNA in the sample can be extracted by more intense methods such as boiling.
  • the specific steps are: take 20 ⁇ L of the fresh sample to be tested, such as fecal sample, and place it in a 2.0 mm FTA filter. The microcentrifuge tube was then dried at 56°C. The dried FTA filter was added with 200 ⁇ l of 10% SDS solution, boiled for 10 min, washed twice with FTA special buffer, and then washed twice with TE buffer, 56 After drying at °C, it can be used as a PCR reaction template.
  • step (3) Add the DNA extracted in step (1) as a template to the PCR-A reaction system and the PCR-B reaction system according to Table 3, perform PCR amplification reaction and perform fluorescent PCR detection, PCR amplification in PCR detection
  • the product is subjected to high-resolution dissolution analysis.
  • the procedure is: 95°C15s, 60°C1min, 95°C15s, 60°C15s, and the dissolution rate is 0.2°C/s; when the PCR-A reaction system and PCR-B reaction system are either When the actual melting curve Tm value of a system matches the standard melting curve Tm value of any type of MCR gene, the sample to be tested has the MCR gene.
  • the PCR amplification reaction program conditions are as follows: 95 °C 2min, 1 cycle; 95 °C 5sec, 60 °C 40sec, 40 cycles.
  • the real-time fluorescence quantitative PCR instrument of Applied Biosystems ABI 7500 manufactured by Thermo Fisher Scientific Co., Ltd. is used for the detection of fluorescent PCR, but it is not limited to this.
  • Select the detection channel of the instrument When performing the fluorescent PCR reaction, the collection of the fluorescent signal of the reaction tube in the instrument used should be set. The specific detection method of the fluorescence detection channel depends on the instrument, and refer to the instruction manual of the instrument.
  • the actual dissolution curve Tm value obtained by the PCR reaction system is consistent with the standard dissolution curve Tm value. It can be understood that the actual dissolution curve Tm value falls within the range of the standard dissolution curve Tm value ⁇ 0.2, which is considered to be consistent.
  • the primer pairs described in Table 1 in Example 1 are selected, and the bacterial culture solution to be tested and the culture solutions of a total of 33 non-target strains are used to extract the genomes of bacteria in samples from various sources using phenol-chloroform method or kit DNA.
  • the standard strains are those purchased or preserved by the Food Safety and Testing Laboratory of South China University of Technology; the isolates are from various farms in Shenzhen, farmers' markets and healthy people's feces, and the strains were identified by the method of Shen, et al. (2018) The presence or absence of MCR in, and the strains were identified by 16S sequencing or mass spectrometry.
  • CMCC China Medical Microorganism Strain Collection Center, Huatuo Road, Biomedical Industrial Base, Daxing District, Beijing No. 31, 102629
  • CGMCC China General Microbial Culture Collection Center, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, 100101
  • CICC Industrial Microbial Culture Collection Management Center, Jiuxianqiao Middle Road, Chaoyang District, Beijing Building 6, Building 24, Post Code: 100015; SZCIQ, Shenzhen Entry-Exit Inspection and Quarantine Bureau, No.
  • strains from ATCC, CMCC, CGMCC, CICC can be purchased.
  • Strains from SZCIQ and ADCPC were respectively donated by these institutions; these strains have been published in the literature "Xing-long Xiao, Li Zhang, Hui Wu, Yi-gang Yu, Yu-qian Tang, Dong-mei Liu, Xiao-feng Li.
  • MCR detection method references "Yingbo, S., Hongwei, Z., Jiao, X., Yongqiang, W., Qijing, Z., &Walsh, TR, et.al. (2016). Anthropogenic and environmental factors associated with highincidenceofmcr-1carriageinhumansacrosschina.NatureMicrobiology.”.
  • the culture medium to be tested contains any one or more of MCR variants (MCR gene variants, ie mcr-1 to mcr-6), a positive amplification curve will be displayed, and the Tm value of the dissolution curve and mcr-1
  • the Tm value of any standard dissolution curve to mcr-6 is consistent.
  • the standard dissolution curve of mcr-1 to mcr-6 is shown in Figure 2a-f; if the culture medium to be tested does not contain MCR variants, there is no amplification signal. It is suggested that the above primer pairs have good sensitivity and specificity.
  • the strains in Table 4 were tested, and the results showed that the strains containing the target gene were detected, while the non-target strains were all negative with no amplification curve, indicating that this method has good specificity.
  • the sample to be tested needs to be identified through the melting curve.
  • Tm value as the interpretation standard, as shown in Figures 2a-f
  • the peak value of the standard dissolution curve corresponding to different types of MCR genes.
  • the genes are mcr-1, mcr-2, mcr-5, mcr-6, mcr-3 and mcr-4, indicating that this method has good resolution.
  • the PCR-A reaction system can obtain a dissolution curve with a T m value of 79.6°C. Due to the difference between mcr-1 and mcr-3 primer interaction exists between the pair of the reaction, T m value of 79.6 deg.] C to generate by-products, and PCR-B reaction system T m values were obtained for the two dissolution profiles of 74.8 deg.] C and 80.1 deg.] C, respectively, and T m (mcr- 4) Consistent with T m (mcr-3), so when mcr-3 and mcr-4 are present at the same time, the detection method of the present invention can simultaneously detect the presence of mcr-3 and mcr-4 in the sample.
  • the detection method of the kit of the present invention can not only effectively detect any one of all MCRs (mcr-1 to mcr-6) in the sample to be tested, but when MCR exists at the same time, it can partially detect multiple MCRs at the same time. Genotype function.
  • the feces described in this embodiment are 112 fresh feces samples from healthy people collected by the Huadazhi feces collection kit, suspended in the preservation solution, and stored at 4°C for no more than one week.
  • the primer pairs in Table 1 in Example 1 were selected, and the total DNA of fecal bacteria extracted using the boiling method described in Example 1 and the DNA extracted using the method of Shen, et al. (2016) described in Example 2
  • the template is used as a different DNA template for PCR detection and comparison.
  • the results showed that the positive rate of Shen, et al. (2018) method was 12.5% (14/112), while the positive rate of this method was 13.4% (15/112). It shows that the present invention adopts the boiling method to extract the DNA in the sample to be tested as a template and has a good detection rate.
  • Figures 2a-f and 3a-c are three parallel experiments. Although the dissolution curves in each figure do not overlap, each dissolution curve is automatically calculated according to the software of the PCR detection instrument The Tm is the same, so it is a normal phenomenon in the art that there are slight differences in the shape of the three melting curves of the three parallel experiments of the same DNA sample.

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Abstract

本发明公开了一种检测MCR基因的试剂盒、检测方法及其应用。该试剂盒包括PCR-A组引物和PCR-B组引物，所述PCR-A组引物包括：SEQ ID No:1和SEQ ID No:2所示的引物对、SEQ ID No:3和SEQ ID No:4所示的引物对、SEQ ID No:5和SEQ ID No:6所示的引物对、SEQ ID No:7和SEQ ID No:8所示的引物对以及SEQ ID No:9和SEQ ID No:10所示的引物对；所述PCR-B组引物包括：SEQ ID No:11和SEQ ID No:12所示的引物对、SEQ ID No:13和SEQ ID No:14所示的引物对。

Description

一种检测MCR基因的试剂盒、检测方法及其应用 技术领域
本发明属于医疗卫生以及微生物检测领域，一种用于检测粪便样本中存在mcr-1至mcr-6任一种的试剂盒、检测方法及其应用。
背景技术
多黏菌素(polymyxin)是发现于多黏类芽孢杆菌(Paenibacilluspolymyxa)培养液中的抗菌性多肽，有A、B、C、D、E等五种，其中多黏菌素E(colistin)和多黏菌素B(PMB)是临床中最常用的两种多黏菌素。由于其存在一定的神经毒性，在临床上的使用受到严格限制，但在亚洲、欧洲和北美，多黏菌素广泛应用于禽畜养殖业中。根据中国CHINET细菌耐药性监测，由于耐碳青霉烯类肠杆菌科细菌(Carbapenem-resistant enterobacteriaceae,CRE)的高感染率，多粘菌素作为最后一道防线得到重视，在对CRE菌株感染的危重患者的治疗中，多粘菌素是挽救疗法策略中最为倚重的抗菌药。
近几年，多粘菌素耐药的肠杆菌科细菌在医学临床不断出现并流行传播，成为全球卫生工作者的关注热点。到目前为止对多粘菌素耐药的CRE涵盖产KPC、NDM、VIM和IMI酶的肠杆菌科细菌。然而近几年在临床、养殖厂、动物性食物、甚至健康人群样本中，均发现对多黏菌素耐药的可转移性耐药性MCR(mobile colistin resistance)基因。其中在2015年的调查中显示，广东省健康人群的肠道菌群中MCR的携带率高达15％，这可能是由于多黏菌素在养殖业中的广泛使用，使得多粘菌素耐药基因通过动物性食品到人群的食物链进入到健康人群体中。最令人担忧的是，mcr-1可与产KPC、NDM和VIM等碳青霉烯酶的CRE同时存在，形成名副其实的“超级细菌”。这就意味着其极大的阻碍了作为抗革兰阴性菌最后一道防线的多粘菌素的使用。
自mcr-1首次报道以来，已在临床和环境样本中多次发现；在比利时的动物来源样本中分离得到含mcr-2(LT598652)的E.coli；在来源于中国的猪肉样本中分离得到含mcr-3(KY924928)的E.coli；在来源于中国的猪肉样本中分离得到含mcr-2(LT598652)的E.coli；在来源与欧洲的猪肉样本中分离得到含mcr-4(MF543359)和mcr-5(KY807921)的E.coli和Salmonella entericaserovarTyphimurium；在来源于英国的猪肉样本中分离得到含mcr-6(MF176240)的Moraxella。此外，各类mcr-1,mcr-3和mcr-4的基因变种也相应在肠杆菌科细菌中被发现。目前全世界范围内共有6类MCR被报道发现，且每个基因可能含有多个基因变异类型，其中mcr-1已发现多达11种基因变异类型。携带mcr-1基因的细菌并不局限于我国，在东南亚、欧洲、美洲和非洲的30多个国家均有发现。mcr-1基因的可转移性、高携带率和大范围分布引起社会极大的关注和不安。再者，由于MCR的广泛存在，特别是在健康人群和动物性食品中的存在，使得针对MCR检测现用的选择性筛菌、质谱鉴定菌种、药敏检测和普通PCR验证的检测程序方法变得十分繁复且低效。
虽然中国发明专利申请CN201810531961，发明名称检测多粘菌素耐药基因的多重PCR引物、试剂盒及检测方法，其通过在mcr-1、mcr-2、mcr-3、mcr-4、mcr-5的基因序列基础上进行引物设计，利用不同PCR产物的大小对相应的耐药基因加以区分，对反应体系及条件加以优化之后，最终确定了一套较为完善的多重PCR反应体系，能够对MCR 相关抗性基因进行较为全面的检测。但是该发明专利申请存在以下缺陷：(1)其采用的普通PCR然后凝胶电泳的方法，很容易造成气溶胶的污染，影响检测结果；(2)其多重引物全部加入到一反应体系中，难以避免各引物之间的相互反应，故产生非特异性扩增，影响多重PCR的检测效率和检测质量；(3)无法对PCR产物进行实时监测，不适应于现代临床检测与耐药性监测和筛查中快速、灵敏和高通量的要求。
发明内容
本发明的首要目的在于，提供一种检测可移动多黏菌素耐药性MCR基因的试剂盒，针对现有技术中鲜少有涵盖所有MCR基因(mcr-1至mcr-6)的分子检测方法，本试剂盒基于已发现的所有MCR的基因序列，设计适用于所有MCR检测的荧光PCR检测方法，本试剂盒特别适合于医疗临床或环境监测场景中，能够快速简便有效的对粪便样本中的任一种MCR基因进行检测预警，对PCR产物进行实时监测，将大大降低监测可移动性多粘菌素耐药基因的人力和物力消耗，简化日常生产生活中大批量的临床样品的抽检与监测。再者，本试剂盒的PCR-A反应体系采用了多量的通用引物对和极少量的特异性引物组合的方法，其中特异性引物特异性结合目标片段并扩增后，由通用引物以初始的扩增片段为模板进行扩增，这样的组合可尽可能减少引物之间的相互反应，提升多重PCR的效率；本试剂盒采用荧光PCR技术作为检测方法，整个反应均在封闭的反应管内进行，避免了PCR－电泳等易于形成气溶胶污染而造成假阳性结果。
本发明的另一目的在于提供所述试剂盒的检测方法。
本发明的又一目的在于提供检测可移动的多黏菌素耐药性MCR基因的试剂盒在临床或环境监测中检测MCR的应用。
本发明的目的通过下述技术方案实现：
一种检测可移动的多黏菌素耐药性MCR基因的试剂盒，其包括PCR-A组引物和PCR-B组引物，所述PCR-A组引物包括：SEQ ID No:1和SEQ ID No:2所示的引物对、SEQ ID No:3和SEQ ID No:4所示的引物对、SEQ ID No:5和SEQ ID No:6所示的引物对、SEQ ID No:7和SEQ ID No:8所示的引物对以及SEQ ID No:9和SEQ ID No:10所示的引物对；所述PCR-B组引物包括：SEQ ID No:11和SEQ ID No:12所示的引物对、SEQ ID No:13和SEQ ID No:14所示的引物对。
在本发明中，所述试剂盒还包括：2×PCR缓冲液、Taq酶、dNTP、MgCl 2、荧光染料EvaGreen以及FTA试纸片、10％SDS溶液、TE缓冲液。
在本发明中，该试剂盒为20～50μL荧光PCR反应体系的试剂盒，各组分及其含量如下：
  PCR-A反应体系 PCR-B反应体系
组分 终浓度 终浓度
2×PCR缓冲液 1× 1×
Mg 2+浓度 2.0mmol/L 2.0mmol/L
dNTPs(含dUTP) 0.15mmol/L 0.15mmol/L
Taq酶 2U 2U
20×EvaGreen荧光染料 1× 1×
SEQ ID No:1所示引物 0.8μmol/L 无
SEQ ID No:2所示引物 0.8μmol/L 无
SEQ ID No:3所示引物 0.05μmol/L 无
SEQ ID No:4所示引物 0.05μmol/L 无
SEQ ID No:5所示引物 0.05μmol/L 无
SEQ ID No:6所示引物 0.05μmol/L 无
SEQ ID No:7所示引物 0.05μmol/L 无
SEQ ID No:8所示引物 0.05μmol/L 无
SEQ ID No:9所示引物 0.05μmol/L 无
SEQ ID No:10所示引物 0.05μmol/L 无
SEQ ID No:11所示引物 无 0.5μmol/L
SEQ ID No:12所示引物 无 0.5μmol/L
SEQ ID No:13所示引物 无 0.5μmol/L
SEQ ID No:14所示引物 无 0.5μmol/L
DNA模板 2μL 2μL
补水至 20～50μL 20～50μL
一种检测可移动的多黏菌素耐药性MCR基因的试剂盒的检测方法，其包括如下步骤：
(1)提取待测样品中的DNA；
(2)分别配置具有PCR-A组引物的PCR-A反应体系和具有PCR-B组引物的PCR-B反应体系；
(3)将步骤(1)提取的DNA作为模板分别加入到PCR-A反应体系和PCR-B反应体系中，进行PCR扩增反应并进行荧光PCR检测；当PCR-A反应体系和PCR-B反应体系中的任一体系的实际溶解曲线Tm值与任一种类MCR基因的标准溶解曲线Tm值相符时，则待测样品具有MCR基因。
在本发明中，步骤(1)具体包括如下步骤：取20μL待测样品，置于有直径2.0mmFTA滤膜片的离心管中，然后56℃干燥，干燥后的FTA滤膜片加入10％SDS溶液200μL，煮沸10min，用FTA专用缓冲液洗涤2次，然后再用TE缓冲液洗涤两次，56℃干燥后，可作为PCR反应模板。
在本发明中，所述试剂盒还包括：2×PCR缓冲液、Taq酶、dNTP、MgCl 2、荧光染料EvaGreen以及FTA试纸片、10％SDS溶液、TE缓冲液。
在本发明中，该试剂盒为20～50μL荧光PCR反应体系的试剂盒，各组分及其含量如下：
  PCR-A反应体系 PCR-B反应体系
组分 终浓度 终浓度
2×PCR缓冲液 1× 1×
Mg 2+浓度 2.0mmol/L 2.0mmol/L
dNTPs(含dUTP) 0.15mmol/L 0.15mmol/L
Taq酶 2U 2U
20×EvaGreen荧光染料 1× 1×
SEQ ID No:1所示引物 0.8μmol/L 无
SEQ ID No:2所示引物 0.8μmol/L 无
SEQ ID No:3所示引物 0.05μmol/L 无
SEQ ID No:4所示引物 0.05μmol/L 无
SEQ ID No:5所示引物 0.05μmol/L 无
SEQ ID No:6所示引物 0.05μmol/L 无
SEQ ID No:7所示引物 0.05μmol/L 无
SEQ ID No:8所示引物 0.05μmol/L 无
SEQ ID No:9所示引物 0.05μmol/L 无
SEQ ID No:10所示引物 0.05μmol/L 无
SEQ ID No:11所示引物 无 0.5μmol/L
SEQ ID No:12所示引物 无 0.5μmol/L
SEQ ID No:13所示引物 无 0.5μmol/L
SEQ ID No:14所示引物 无 0.5μmol/L
DNA模板 2μL 2μL
补水至 20～50μL 20～50μL
一种检测可移动的多黏菌素耐药性MCR基因的试剂盒在临床或环境监测中检测MCR的应用。
本发明相对于现有技术具有如下的优点及效果：
(1)针对现有技术中鲜少有涵盖所有MCR基因(mcr-1至mcr-6)的分子检测方法，本试剂盒基于已发现的所有MCR的基因序列，设计适用于所有MCR检测的荧光PCR检测方法。
(2)本试剂盒特别适合于医疗临床或环境监测场景中，能够快速简便有效的对粪便样本中的任一种MCR基因进行检测预警，对PCR产物进行实时监测，将大大降低监测可移动性多粘菌素耐药基因的人力和物力消耗，简化日常生产生活中大批量的临床样品的抽检与监测，因此本试剂盒及其检测方法在医疗临床及环境监测场景中具有较大的应用价值。目前常规shen,et al2018(实施例2有提到)的方法，对环境和人群中的mcr-1和mcr-3进行监测，其流程包括选择性筛菌、质谱鉴定菌种、药敏检测和PCR验证，总时长需5～7天；而本方法可减少选择性筛菌步骤，直接从环境总DNA中检测目标基因，时间可缩短至6～24h。
(3)本试剂盒的PCR-A反应体系采用了多量的通用引物对和极少量的特异性引物组合的方法，其中特异性引物特异性结合目标片段并扩增后，由通用引物以初始的扩增片段为模板进行扩增，这样的组合可尽可能减少引物之间的相互反应，提升多重PCR的效率。
(4)本试剂盒采用荧光PCR技术作为检测方法，整个反应均在封闭的反应管内进行，避免了PCR－电泳等易于形成气溶胶污染而造成假阳性结果。
(5)本发明提供的引物对于不含有MCR的检测样本均无扩增信号，说明其具有良好的特异性。
附图说明
图1为对NCBI数据库中46条MCR基因的SeqMan序列分析图。
图2a为mcr-1的标准溶解曲线，包括三条阳性模板的溶解曲线，Tm(mcr-1)＝84.8℃；
图2b为mcr-2的标准溶解曲线，包括三条阳性模板的溶解曲线，Tm(mcr-2)＝85.2℃；
图2c为mcr-5的标准溶解曲线，包括三条阳性模板的溶解曲线，Tm(mcr-5)＝84.1℃；
图2d为mcr-6的标准溶解曲线，包括三条阳性模板的溶解曲线，Tm(mcr-6)＝83.3℃；
图2e为mcr-3的标准溶解曲线，包括三条阳性模板的溶解曲线，Tm(mcr-3)＝80.7℃；
图2f为mcr-4的标准溶解曲线，包括三条阳性模板的溶解曲线，Tm(mcr-4)＝74.8℃；
图3a-A和3a-B为具有mcr-1与mcr-3的混合物样本的溶解曲线，其中3a-A为PCR-A反应体系得到的溶解曲线，3a-B为PCR-B反应体系得到的溶解曲线；
图3b-A和3b-B为具有mcr-2、mcr-5与mcr-6的混合物样本的溶解曲线，其中3b-A为PCR-A反应体系得到的溶解曲线，3b-B为PCR-B反应体系得到的溶解曲线；
图3c-A和3c-B为具有mcr-3与mcr-4的混合物样本的溶解曲线，其中3c-A为PCR-A反应体系得到的溶解曲线，3c-B为PCR-B反应体系得到的溶解曲线。
其中溶解曲线图的纵坐标为Derivative导数，具体为荧光强度的变化对温度的变化求导，即d(Fluorescence)/d(T)或d(荧光值)/d(温度)；横坐标为Temperature温度(℃)，即溶解过程的每一个温度点。峰值处为溶解温度Tm。
具体实施方式
下面结合实施例和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
1、本发明试剂盒中的引物序列设计：通过分别对所有已知的MCR基因序列进行分析，mcr-1至mcr-5均位于质粒或组基因，mcr-6位于组基因，对共46条基因采用SeqMan进行序列分析(图1)，通过分析，找出高度保守的区段，通过premier 3.0选择无二级结 构且高度保守的区段，设计多组引物，引物长度一般为20个碱基左右，引物间和引物内无互补序列。根据预实验结果，mcr-1的引物对与mcr-3的引物之间存在交叉反应，能够互为模板进行扩增，扩增片段T m值约为79.9℃，因此对为避免引物之间反应，将引物分为A和B两组，引物列表1如下所示：
表1引物与扩增片段序列列表(均为5'-3')
Figure PCTCN2019074897-appb-000001
2、反应体系的建立和优化：反应体系的建立和优化中所采用的靶区域模板以如下方法获得：分别取mcr-1至mcr-6携带株的大肠杆菌和肺炎克雷伯氏菌菌株复苏后培养48小时，取培养液1mL经过无菌水洗涤后复溶到1mL，采用酚-氯仿法或试剂盒分别提取基因组核酸，再用上述引物进行PCR扩增，并取其中Ct值24-27之间者作为以后反应体系优化时的模板。其中最初反应体系(40μL)如表2所示：
表2最初设计的PCR反应体系
  PCR-A反应体系 PCR-B反应体系
组分 终浓度 终浓度
2×PCR反应缓冲液 1× 1×
Mg 2+浓度 2.0mmol/L 2.0mmol/L
dNTPs(含dUTP) 0.15mmol/L 0.15mmol/L
Taq酶 2U 2U
20×EvaGreen荧光染料 1× 1×
SEQ ID No:1所示引物 0.5μmol/L 无
SEQ ID No:2所示引物 0.5μmol/L 无
SEQ ID No:3所示引物 0.05μmol/L 无
SEQ ID No:4所示引物 0.05μmol/L 无
SEQ ID No:5所示引物 0.05μmol/L 无
SEQ ID No:6所示引物 0.05μmol/L 无
SEQ ID No:7所示引物 0.05μmol/L 无
SEQ ID No:8所示引物 0.05μmol/L 无
SEQ ID No:9所示引物 0.05μmol/L 无
SEQ ID No:10所示引物 0.05μmol/L 无
SEQ ID No:11所示引物 无 0.8μmol/L
SEQ ID No:12所示引物 无 0.8μmol/L
SEQ ID No:13所示引物 无 0.8μmol/L
SEQ ID No:14所示引物 无 0.8μmol/L
模板 2μL 2μL
补水至 40μL 40μL
2.1引物浓度的优化：在反应体系中，将引物浓度分别从0.1μmol/L～0.8μmol/L作倍比连续稀释后进行检测，通过试验结果的分析比较，确定最佳引物终浓度为反应A：通用引物(SEQ ID No:1和SEQ ID No:2所示引物)浓度为0.8μmol/L，特异性引物(SEQ ID No:3至SEQ ID No:10所示引物)浓度为0.05μmol/L；反应B：特异性引物(SEQ ID No:11至SEQ ID No:14所示引物)浓度为0.5μmol/L。
2.2镁离子浓度的优化：在引物的终浓度为0.2μmol/L以及反应体系中其它条件同表2的前提下，将MgCl 2的浓度从1mmol/L～2.5mmol/L以0.5mmol/L递增，经过多次重复实验选定2.5mmol/L为试剂盒反应体系中的镁离子浓度。
2.3 Taq DNA聚合酶(Taq酶)用量的优化：在引物的终浓度为0.2μmol/L、镁离子的终浓度为2.5mmol/L以及反应体系中其它条件同表2的前提下，通过比较Taq酶用量(以单位Unit计)的优化实验结果，选定2U作为试剂盒反应体系中Taq酶的用量。
2.4 dNTPs(脱氧核糖核苷三磷酸)浓度的优化：在引物的终浓度为0.2μmol/L、镁离子的终浓度为2.5mmol/L以及反应体系中其它条件同表2的前提下，通过使用不同浓度的dNTPs进行检测，综合评估后选0.2mmol/L作为试剂盒反应体系中dNTPs的使用量。
由于本法所涉及的引物及目标片段众多，非饱和染料SYBRGREEN误差较大，无法区分单基因差异，因此本法采用饱和染料EvaGreen作为PCR过程中的荧光染料。Evagreen对qPCR干扰极小，无“染料重分布”，饱和性嵌入DNA双链，分辨效果远优于SYBR Green。利用上述引物及荧光染料进行反应体系的建立，最后确定采用的荧光PCR反应体系为40μL体系，所需各组分及相应浓度见表3。
表3优化后的PCR反应体系
  PCR-A反应体系 PCR-B反应体系
组分 终浓度 终浓度
2×PCR反应缓冲液 1× 1×
Mg 2+浓度 2.0mmol/L 2.0mmol/L
dNTPs(含dUTP) 0.15mmol/L 0.15mmol/L
Taq酶 2U 2U
20×EvaGreen荧光染料 1× 1×
SEQ ID No:1所示引物 0.8μmol/L 无
SEQ ID No:2所示引物 0.8μmol/L 无
SEQ ID No:3所示引物 0.05μmol/L 无
SEQ ID No:4所示引物 0.05μmol/L 无
SEQ ID No:5所示引物 0.05μmol/L 无
SEQ ID No:6所示引物 0.05μmol/L 无
SEQ ID No:7所示引物 0.05μmol/L 无
SEQ ID No:8所示引物 0.05μmol/L 无
SEQ ID No:9所示引物 0.05μmol/L 无
SEQ ID No:10所示引物 0.05μmol/L 无
SEQ ID No:11所示引物 无 0.5μmol/L
SEQ ID No:12所示引物 无 0.5μmol/L
SEQ ID No:13所示引物 无 0.5μmol/L
SEQ ID No:14所示引物 无 0.5μmol/L
模板 2μL 2μL
补水至 40μL 40μL
注：在荧光PCR反应体积不同如20～50μL时，各试剂应按比例调整。
具体地，以表3为例，则：
PCR-A反应体系为：每40μL PCR体系包括2×PCR缓冲液20μL；浓度为5U/μL的Taq酶0.4μL；浓度为10mmol/L的dNTP 4μL；浓度为25mmol/L的MgCl 23.2μL；浓度为10μmol/L的SEQ ID No:1所示引物和SEQ ID No:2所示引物各3.2μL，10μmol/L的SEQ ID No:3所示引物、SEQ ID No:4所示引物、SEQ ID No:5所示引物、SEQ ID No:6所示引物、SEQ ID No:7所示引物、SEQ ID No:8所示引物、SEQ ID No:9所示引物和SEQ ID No:10所示引物各0.2μL，模板DNA 2μL；荧光染料20×EvaGreen 2μL；用去核酸水补齐至40μL；
PCR-B反应体系为：每40μL PCR体系包括2×PCR缓冲液20μL；浓度为5U/μL的Taq酶0.4μL；浓度为10mmol/L的dNTP 4μL；浓度为25mmol/L的MgCl 23.2μL；浓度为10μmol/L的SEQ ID No:11所示引物、SEQ ID No:12所示引物、SEQ ID No:13所示引物和SEQ ID No:14所示引物各2μL，模板DNA 2μL；荧光染料20×EvaGreen 2μL；用去核酸水补齐至40μL。
3、本发明试剂盒的检测方法，具体包括以下步骤：
(1)提取待测样品中的DNA；
由于已知的可移动MCR均在质粒上，因此可用煮沸法等较为激烈的方法提取样本中的DNA，具体步骤为：取20μL新鲜的待测样品如粪便样本，置于有直径2.0mmFTA滤膜片的离心管中，然后56℃干燥，干燥后的FTA滤膜片加入10％SDS溶液200微升，煮沸10min，用FTA专用缓冲液洗涤2次，然后再用TE缓冲液洗涤两次，56℃干燥后，可作为PCR反应模板。
(2)按表3分别配置具有PCR-A组引物的PCR-A反应体系和具有PCR-B组引物的PCR-B反应体系；
(3)将步骤(1)提取的DNA作为模板按表3分别加入到PCR-A反应体系和PCR-B反应体系中，进行PCR扩增反应并进行荧光PCR检测，PCR检测中对PCR扩增产物进行高分辨率溶解分析，程序为：95℃15s，60℃1min，95℃15s，60℃15s，溶解速率为0.2℃/s；当PCR-A反应体系和PCR-B反应体系中的任一体系的实际溶解曲线Tm值与任一种类MCR基因的标准溶解曲线Tm值相符时，则待测样品具有MCR基因。
其中，PCR扩增反应程序条件如下：95℃2min，1个循环；95℃5sec、60℃40sec，40个循环。
本实施例中荧光PCR检测使用赛默飞世尔科技有限公司产的Applied Biosystems ABI 7500型的实时荧光定量PCR仪，但不局限于此。选择仪器的检测通道：在进行荧光PCR反应时，应对所用仪器中反应管荧光信号的收集进行设置，选择的荧光检测通道，具体设置方法因仪器而异，应参照仪器使用说明书。
需要说明的是，PCR反应体系得到的实际溶解曲线Tm值与标准溶解曲线Tm值相符，可以理解为，实际溶解曲线Tm值落入在标准溶解曲线Tm值±0.2的范围内即视为相符。
实施例2
选取实施例1中表1的所述引物对，将待检的细菌培养液和其他非目标菌株共33株菌的培养液用采用酚-氯仿法或试剂盒提取各种来源样品中细菌的基因组DNA。其中标准菌株为华南理工大学食品安全与检测实验室购买或保藏菌株；分离株来源于深圳市各养殖场、农贸市场与健康人群粪便中，并采用Shen,et al.(2018)的方法鉴定菌株中有无MCR的存在，且所述菌株均经过16S测序或质谱鉴定。
在40μL荧光PCR反应体系(按表3配制)中，加入以上提取的不同菌株的基因组DNA 2μL，根据实施例1中的PCR反应条件进行荧光PCR检测。
实验结果如下表4所示。
表4不同菌株的荧光定量qPCR结果
Figure PCTCN2019074897-appb-000002
Figure PCTCN2019074897-appb-000003
a.+，阳性；-，阴性；
b.ATCC，美国菌种保藏中心，美弗吉尼亚洲马纳萨斯镇丹佛大学美国菌种保藏中心，邮编10801；CMCC，中国医学微生物菌种保藏中心，北京市大兴区生物医药产业基地华佗路31号院，邮编102629；CGMCC，中国普通微生物菌种保藏中心，北京市朝阳区北辰西路1号院3号，邮编100101；CICC，工业微生物菌种保藏管理中心，北京市朝阳区酒仙桥中路24号院6号楼，邮编：100015；SZCIQ，深圳出入境检验检疫局，广东省深圳市福田区福强路1011号，邮编518000；ADCPC，重庆动物疾病控制和预防中心，重庆市渝中区长江二路8号，邮编：400042。其中来自于ATCC、CMCC、CGMCC、CICC的菌种均可以购买得到。来自于SZCIQ与ADCPC的菌株由这些机构分别惠赠；这些菌株已在文献“Xing-long Xiao,Li Zhang,Hui Wu,Yi-gang Yu,Yu-qian Tang,Dong-mei Liu,Xiao- feng Li.Simultaneous Detection of Salmonella,Listeriamonocytogenes,andStaphylococcusaureus by Multiplex Real-Time PCR Assays Using High-Resolution Melting[J].Food Analytical Methods,2014,7(10):1960-1972”中公开；
c.MCR检测方法参考文献“Yingbo,S.,Hongwei,Z.,Jiao,X.,Yongqiang,W.,Qijing,Z.,&Walsh,T.R.,et al.(2018).Anthropogenic and environmental factors associated with high incidence of mcr-1carriage in humans across china.Nature Microbiology.”。
经检测，若待检培养液中含有MCR variants(MCR的基因变异型，即mcr-1至mcr-6)中任意一种或以上则显示阳性扩增曲线，且溶解曲线Tm值与mcr-1至mcr-6任一标准溶解曲线的Tm值相符，其中mcr-1至mcr-6的标准溶解曲线如图2a-f所示；若待检培养液中不含有MCR variants则无扩增信号，提示上述引物对具有良好的灵敏度和特异性。对表4中的菌株进行检测，结果表明含目标基因的菌株均有检出，而非目标菌株均为无扩增曲线为阴性，说明本法具有良好的特异性。
进一步地，为了确定待测样品中的MCR种类，则需要通过熔解曲线对待测样本进行鉴定。使用Tm值作为判读标准，如图2a-f所示不同种类MCR基因对应的标准溶解曲线的峰值，当测得的Tm值为84.8、85.2、84.1、83.3、80.7和74.8时，则对应耐药基因分别为mcr-1、mcr-2、mcr-5、mcr-6、mcr-3和mcr-4，说明本法具有良好的分辨率。
具体实际检测过程中，由于不同种类MCR基因可能同时出现，因此进一步对混合DNA模板进行了检测，其中试验所述混合样本均为等比例混合的DNA模板，结果如图3a-c所示。
如图3a-A和3a-B所示，当mcr-1与mcr-3同时存在时，PCR-A反应体系可得到T m值为84.8℃的溶解曲线，与T m(mcr-1)一致，而PCR-B反应体系可得到T m值为79.6℃的溶解曲线，这是由于mcr-1与mcr-3的引物对之间存在交互反应，生成T m值为79.6℃副产物，说明当mcr-1与mcr-3同时存在时，本发明检测方法能测得mcr-1的存在，无法推测mcr-3是否存在。
如图3b-A和3b-B所示，当mcr-2、mcr-5以及mcr-6同时存在时，PCR-A反应体系可得到T m值为84.1℃的溶解曲线，与T m(mcr-5)一致，这可能是由于mcr-5的引物与底物具有更强的亲和力，而PCR-B反应体系无明显特征的溶解曲线，说明当mcr-2、mcr-5以及mcr-6同时存在时，本发明检测方法至少可测得mcr-5的存在。
如图3c-A和3c-B所示，当mcr-3与mcr-4同时存在时，PCR-A反应体系可得到T m值为79.6℃的溶解曲线，由于mcr-1与mcr-3的引物对之间存在交互反应，生成T m值为79.6℃副产物，而PCR-B反应体系中可得到T m值分别为74.8℃和80.1℃的两个溶解曲线，分别与T m(mcr-4)和T m(mcr-3)一致，因此当mcr-3与mcr-4同时存在时，本发明检测方法可同时测得样本中mcr-3和mcr-4的存在。
综上，本发明试剂盒检测方法不仅可有效的检出待测样本中的所有MCR(mcr-1至 mcr-6)的任一种，当MCR同时存在时，具有部分同时检出多个MCR基因型的功能。
实施例3
本实施例所述粪便为采用华大智造粪便采集套装采集的健康人群新鲜粪便样本共112份，悬浮于保存液中，并置于4℃保存不超过一周的粪便样本。
选取实施例1中表1的引物对，将使用实施例1所述煮沸法所提取到的粪便细菌总DNA和采用实施例2中所述Shen,et al.(2018)的方法提取到的DNA模板作为不同的DNA模板进行PCR检测对比。结果表明，Shen,et al.(2018)的方法检出阳性率为12.5％(14/112)，而本法检出阳性率为13.4％(15/112)。说明本发明采用煮沸法提取待测样品中的DNA作为模板具有良好的检出率。
需要说明的是：图2a-f和图3a-c，均为3次平行实验，从每张图中的溶解曲线虽然看起并未重合，但每条溶解曲线根据PCR检测仪器的软件自动算出来的Tm均是一样的，故同一DNA样本进行3次平行实验的3次溶解曲线形态存在细微差别属于本领域正常现象。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。
自由核苷酸系列
序列表
Figure PCTCN2019074897-appb-000004
Figure PCTCN2019074897-appb-000005
Figure PCTCN2019074897-appb-000006

Claims (8)

  1. 一种检测MCR基因的试剂盒，其特征在于，其包括PCR-A组引物和PCR-B组引物，所述PCR-A组引物包括：SEQ ID No:1和SEQ ID No:2所示的引物对、SEQ ID No:3和SEQ ID No:4所示的引物对、SEQ ID No:5和SEQ ID No:6所示的引物对、SEQ ID No:7和SEQ ID No:8所示的引物对以及SEQ ID No:9和SEQ ID No:10所示的引物对；所述PCR-B组引物包括：SEQ ID No:11和SEQ ID No:12所示的引物对、SEQ ID No:13和SEQ ID No:14所示的引物对。
  2. 根据权利要求1所述的检测MCR基因的试剂盒，其特征在于，还包括：2×PCR缓冲液、Taq酶、dNTP、MgCl 2、荧光染料EvaGreen以及FTA试纸片、10％SDS溶液、TE缓冲液。
  3. 根据权利要求2所述的检测MCR基因的试剂盒，其特征在于，该试剂盒为20～50μL荧光PCR反应体系的试剂盒，各组分及其含量如下：
      PCR-A反应体系 PCR-B反应体系 组分 终浓度 终浓度 2×PCR缓冲液 1× 1× Mg 2+浓度 2.0mmol/L 2.0mmol/L dNTPs(含dUTP) 0.15mmol/L 0.15mmol/L Taq酶 2U 2U 20×EvaGreen荧光染料 1× 1× SEQ ID No:1所示引物 0.8μmol/L 无 SEQ ID No:2所示引物 0.8μmol/L 无 SEQ ID No:3所示引物 0.05μmol/L 无 SEQ ID No:4所示引物 0.05μmol/L 无 SEQ ID No:5所示引物 0.05μmol/L 无 SEQ ID No:6所示引物 0.05μmol/L 无 SEQ ID No:7所示引物 0.05μmol/L 无 SEQ ID No:8所示引物 0.05μmol/L 无 SEQ ID No:9所示引物 0.05μmol/L 无 SEQ ID No:10所示引物 0.05μmol/L 无 SEQ ID No:11所示引物 无 0.5μmol/L SEQ ID No:12所示引物 无 0.5μmol/L SEQ ID No:13所示引物 无 0.5μmol/L SEQ ID No:14所示引物 无 0.5μmol/L DNA模板 2μL 2μL 补水至 20～50μL 20～50μL
    。
  4. 一种如权利要求1所述的检测MCR基因的试剂盒的检测方法，其特征在于，包括如下步骤：
    (1)提取待测样品中的DNA；
    (2)分别配置具有PCR-A组引物的PCR-A反应体系和具有PCR-B组引物的PCR-B反应体系；
    (3)将步骤(1)提取的DNA作为模板分别加入到PCR-A反应体系和PCR-B反应体系中， 进行PCR扩增反应并进行荧光PCR检测；当PCR-A反应体系和PCR-B反应体系中的任一体系的实际溶解曲线Tm值与任一种类MCR基因的标准溶解曲线Tm值相符时，则待测样品具有MCR基因。
  5. 根据权利要求4所述的试剂盒的检测方法，其特征在于，步骤(1)具体包括如下步骤：取20μL待测样品，置于有直径2.0mmFTA滤膜片的离心管中，然后56℃干燥，干燥后的FTA滤膜片加入10％SDS溶液200μL，煮沸10min，用FTA专用缓冲液洗涤2次，然后再用TE缓冲液洗涤两次，56℃干燥后，可作为PCR反应模板。
  6. 根据权利要求4所述的试剂盒的检测方法，其特征在于，所述试剂盒还包括：2×PCR缓冲液、Taq酶、dNTP、MgCl 2、荧光染料EvaGreen以及FTA试纸片、10％SDS溶液、TE缓冲液。
  7. 根据权利要求4或5所述的试剂盒的检测方法，其特征在于，该试剂盒为20～50μL荧光PCR反应体系的试剂盒，各组分及其含量如下：
      PCR-A反应体系 PCR-B反应体系 组分 终浓度 终浓度 2×PCR缓冲液 1× 1× Mg 2+浓度 2.0mmol/L 2.0mmol/L dNTPs(含dUTP) 0.15mmol/L 0.15mmol/L Taq酶 2U 2U 20×EvaGreen荧光染料 1× 1× SEQ ID No:1所示引物 0.8μmol/L 无 SEQ ID No:2所示引物 0.8μmol/L 无 SEQ ID No:3所示引物 0.05μmol/L 无 SEQ ID No:4所示引物 0.05μmol/L 无 SEQ ID No:5所示引物 0.05μmol/L 无 SEQ ID No:6所示引物 0.05μmol/L 无 SEQ ID No:7所示引物 0.05μmol/L 无 SEQ ID No:8所示引物 0.05μmol/L 无 SEQ ID No:9所示引物 0.05μmol/L 无 SEQ ID No:10所示引物 0.05μmol/L 无 SEQ ID No:11所示引物 无 0.5μmol/L SEQ ID No:12所示引物 无 0.5μmol/L SEQ ID No:13所示引物 无 0.5μmol/L SEQ ID No:14所示引物 无 0.5μmol/L DNA模板 2μL 2μL 补水至 20～50μL 20～50μL
    。
  8. 一种如权利要求1至3任一所述的检测MCR基因的试剂盒在临床或环境监测中检测MCR的应用。
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