A high-throughput detection primer set, chip, kit and detection method for microbial antibiotic resistance genes

Abstract

The present invention discloses a high-throughput detection primer set, chip, kit and detection method of a microbial antibiotic resistance gene. The primer set contains 101 pairs of primer sequences, wherein SEQ ID NO: 1 and 2 are a pair of upstream and downstream primers, SEQ ID NO: 3 and 4 are a pair of upstream and downstream primers, and so on until SEQ ID NO: 201 and 202 are a pair of upstream and downstream primers. The present invention not only retains the high sensitivity and accuracy of fluorescence quantitative PCR detection, but also has the advantages of high-throughput reaction of chip detection, thereby reducing the detection cost; the present invention can simultaneously detect 145 resistance genes with 101 pairs of primers, which is beneficial to the daily monitoring and risk assessment of environmental resistance genes.

Description

Technical Field

[0001] The invention relates to gene detection, and in particular to a high-throughput detection primer set, a chip, a kit and a detection method thereof for microbial antibiotic resistance genes. Background Art

[0002] The main risk of antibiotic resistance genes (also referred to as resistance genes) in the environment is that they can be transmitted to human pathogens through horizontal gene transfer, forming new or multi-resistant pathogens, thereby affecting the efficacy of antibiotics and endangering human health. At present, a variety of resistance genes have been detected in multiple environments such as nature, engineering and clinical, and the abundance of resistance genes varies greatly between different environmental media.

[0003] The commonly used fluorescent quantitative PCR method is difficult to achieve rapid detection of multiple resistance genes and a large number of samples due to throughput limitations. Given that environmental samples are usually characterized by complex pollution, if multiple resistance genes need to be quantified, the workload required is large, time-consuming, and costly. Therefore, it is necessary to develop a high-throughput quantitative PCR detection technology that can simultaneously quantify multiple resistance genes, and strive to achieve efficient and rapid detection of resistance genes in the environment, thereby providing an effective basis for accurately assessing the risk and regulation of resistance genes in complex environmental sample systems. Summary of the invention

[0004] The purpose of the present invention is to provide a primer set for a high-throughput quantitative detection method of resistance genes in an environment, which can be used to efficiently, quickly and accurately quantify the abundance of microbial resistance genes and has the characteristics of high sensitivity, strong specificity and wide coverage.

[0005] To achieve the above object, the present invention provides a primer set for high-throughput detection of microbial antibiotic resistance genes, characterized in that the primer set contains 101 pairs of primer sequences, wherein SEQ ID NO: 1 and 2 are a pair of upstream and downstream primers, SEQ ID NO: 3 and 4 are a pair of upstream and downstream primers, and so on to SEQ ID NO: 201 and 202 are a pair of upstream and downstream primers.

[0006] The present invention also provides a high-throughput quantitative detection chip for microbial resistance genes, characterized in that it contains the primer set.

[0007] Furthermore, it also contains positive clone plasmids; the positive plasmid is a plasmid with a resistance gene inserted, and the resistance gene is any gene amplified by the primer set. Each positive plasmid corresponds to a gene.

[0008] The present invention also provides a high-throughput quantitative detection kit for microbial resistance genes, characterized in that it contains the primer set.

[0009] Furthermore, it also contains a positive clone plasmid; the positive plasmid is a plasmid with a resistance gene inserted, and the resistance gene is any gene amplified by the primer set.

[0010] The primer set, the detection chip or the detection kit is used for detecting microbial resistance genes in environmental samples.

[0011] The present invention also provides a high-throughput quantitative detection method for microbial resistance genes, characterized in that it comprises the following steps:

[0012] Extracting total DNA from samples to be tested;

[0013] Amplification: using the primer set or the detection chip or the detection kit to perform PCR amplification on the sample to be tested;

[0014] Result analysis: The abundance of microbial resistance genes was absolutely quantified using the standard plasmid external standard method.

[0015] Furthermore, the PCR amplification procedure is: pre-denaturation at 95°C for 10 min; followed by denaturation at 95°C for 30 s, annealing and extension at 60°C for 30 s, and 40 cycles.

[0016] The present invention designs 101 pairs of primers based on 145 resistance genes (see Table 1). These 145 resistance genes have a resistance gene enrichment multiple higher than 100 in human-related environments and are located on mobile genetic elements. In the primer sequences shown in Table 1, SEQ ID NO: 1 and 2 are a pair of upstream and downstream primers, SEQ ID NO: 3 and 4 are a pair of upstream and downstream primers, and so on to SEQ ID NO: 201 and 202 are a pair of upstream and downstream primers.

[0017] Table 1 Primer related information

[0018]

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025] Label: MLS: macrolide-lincosamide-streptogramin; Y in SEQ ID NO: 204 represents cor t / u.

[0026] The present invention not only retains the high sensitivity and accuracy of fluorescence quantitative PCR detection, but also has the advantages of high-throughput reaction of chip detection, thereby reducing the detection cost;

[0027] The present invention uses a standard plasmid external standard method to absolutely quantify the abundance of microbial resistance genes, and the detection accuracy is high;

[0028] The present invention can detect 145 resistance genes simultaneously using 101 pairs of primers, which is conducive to more comprehensive and accurate assessment of the risks of environmental resistance genes.

[0029] The present invention provides a favorable high-throughput detection method for characterizing environmental microbial resistance groups, which is beneficial to the daily monitoring and risk assessment of environmental resistance genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a diagram showing the distribution scheme of resistance gene primers and control (16S rRNA) in 384-well plates during chip preparation.

[0031] Figure 2 It is a diagram of the distribution scheme of the standard curve plasmid sample in Example 1, the DNA sample from the sewage treatment plant and the sterile water as the negative control in a 384-well plate.

[0032] Figure 3 This is the standard curve of high-throughput fluorescence quantification of 101 primer pairs.

[0033] Figure 4 The sensitivity of 101 primer pairs was evaluated by serial dilution of mixed plasmid.

[0034] Figure 5 It is the amplification efficiency graph of 101 primer pairs.

[0035] Figure 6 This is a graph showing the absolute abundance of resistance genes in Example 1. DETAILED DESCRIPTION

[0036] Embodiments of the present invention are described in detail below, and the examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions from beginning to end. The embodiments described below by reference to the accompanying drawings are exemplary, are intended to be used to explain the present invention, and are not to be construed as limitations of the present invention. In the embodiments, those without specifying specific techniques or conditions are carried out according to the techniques or conditions described in the documents in this area or according to the product specification. Those without specifying the manufacturer of reagents used or instruments are all conventional products that can be obtained commercially.

[0037] Embodiment 1:

[0038] The high-throughput detection method of the present invention is further described by taking samples from a sewage treatment plant as an example.

[0039] 1. Description of sewage treatment plant water samples

[0040] Water samples were collected from the sewage treatment plant of Guangzhou Baiyunshan Pharmaceutical Factory, including the high-concentration antibiotic inlet section (INF-H), low-concentration antibiotic inlet section (INF-L), aerobic pool (OT), anaerobic pool (AT) and effluent section (EFF). In order to eliminate the influence of weather factors on the samples, the samples were collected on days without rainfall before and after the collection.

[0041] 2. Extraction of sample DNA

[0042] The five sewage samples collected were filtered using a 0.22 μm filter membrane, and the filter membrane was cut into pieces. Total DNA was extracted using a kit from MP Biopharmaceuticals (USA). The extracted DNA was subjected to electrophoresis on a 1% agarose gel to detect the extraction effect. The concentration of the extracted DNA was determined using a Qubit assay kit (Thermo Scientific, USA) to ensure that the DNA concentration of all samples was 20 ng / μL-40 ng / μL to meet the requirements of subsequent quantitative experiments.

[0043] 3. Preparation of standard curve plasmid samples

[0044] The resistance gene corresponding to each pair of primers is amplified by PCR from the environmental mixed DNA sample, the target fragment is cut and recovered, the DNA is purified, and the PMD is connected. TM 19-T vector, transformation culture, single colony sequencing, Blast comparison verification and plasmid extraction kit (Tiangen, Beijing) were used to obtain the corresponding positive plasmid. The Qubit detection kit was used to determine the concentration of positive plasmids to ensure that the copy number of each positive plasmid was 10 10 copy / μL. Mix all positive plasmids to make a mixed standard solution. Finally, the concentration of the mixed standard solution is 10 8The mixed plasmid was diluted 10-fold to a copy number range of 10 2 -10 8 copies / μL, used to prepare the standard curve.

[0045] 4. Chip Preparation

[0046] The resistance gene primers, five kinds of DNA samples from sewage treatment plants and positive clone plasmids were distributed on the SmartChip nanochip (WaferGen, USA) using the SmartChip multi-sample nanodispenser (WaferGen, USA). The resistance gene primers, DNA samples and positive clone plasmids were distributed on the chip using the 120 primers × 42 samples protocol in the SmartChip multi-sample nanodispenser instrument instructions. First, 911 μL LightCycler480 DNA SYBRGreenI Mix and 547 μL sterile water were mixed to form PCR reaction solution I. According to the instructions of SmartChip multi-sample nanodispenser, 10.9 μL PCR reaction solution I and 2.7 μL resistance gene primers in 96 sterile well plates were loaded into 120 wells of enzyme-free well plates. Then, the primers were sampled from 384 well plates onto the chip (such as Figure 1 , Figure 1 384 plates of resistance genes corresponding primers table). Next, 493 μL LightCycler480 DNA SYBR GreenI Mix, 9 μL sterilized 50 mg / mL bovine serum albumin solution and 287 μL sterile water were mixed to form PCR reaction solution II. According to the instructions of SmartChip multi-sample nanodispenser, 42 wells of the enzyme-free plate were loaded with 15.2 μL PCR reaction solution II and 3.8 μL DNA samples (including standard curve plasmid samples, sewage treatment plant samples and sterile water as negative control), and each sample contained 3 technical replicates. The SmartChip multi-sample nanodispenser was used to distribute the samples from the 384-well plate to the chip loaded with primers (such as Figure 2 ), Figure 2 Note: H2, H3, H4, H5, H6, H7 and H8 represent: copy number is 10 2 Mixed plasmid sample with a copy number of 10 copies / μL 3 Mixed plasmid sample with a copy number of 10 copies / μL 4 Mixed plasmid sample with a copy number of 10 copies / μL 5 Mixed plasmid sample with a copy number of 10 copies / μL 6Mixed plasmid sample with a copy number of 10 copies / μL 7 copies / μL of mixed plasmid samples and a copy number of 10 8 The mixed plasmid samples were 1000 copies / μL, INF-H was the high-concentration antibiotic inlet sample of the sewage treatment plant, INF-L was the low-concentration antibiotic inlet sample, OT was the aerobic pool sample, AT was the anaerobic pool sample, EFF was the effluent sample, and Water was the negative control. After the primers and DNA samples were distributed, the chip needed to be centrifuged (3200rpm / 5min), and the chip after centrifugation was filmed and used for later use.

[0047] 5. High-throughput fluorescence quantitative PCR

[0048] The prepared chip was tested by fluorescence quantitative PCR using SmartChip real-time PCR system. The PCR amplification program was: 95℃ pre-denaturation for 10 min; followed by 95℃ denaturation for 30 s, 60℃ annealing and extension for 30 s, 40 cycles. The program automatically raised the temperature for melting curve analysis.

[0049] 6. Data Analysis

[0050] After the program is completed, the software will automatically analyze and export the data. The exported data is screened according to the following conditions: 1) the amplification efficiency must be between 80% and 120%; 2) the critical cycle value (C T ) must be less than 31; 3) Amplification was considered effective if at least two of the three technical replicates obtained amplification.

[0051] 101 plasmids were used as positive controls for standard curve analysis ( Figure 3 ) and serial dilution assay ( Figure 4 ) to evaluate the specificity, sensitivity and efficiency of 101 primer pairs. Based on the standard curve and the ct value obtained from the experiment, Table 2 was calculated, which is the absolute abundance data of each gene. Figure 6 The absolute abundance sum of resistance genes in Example 1 is shown in FIG. Figure 3 This is the standard curve of high-throughput fluorescence quantification of 101 primer pairs. Figure 4 This is a graph of the sensitivity of 101 primer pairs evaluated by serial dilution of mixed plasmid. Figure 4 It can be seen that the lowest abundance is 0.00028ng / ul. Figure 5 It is the amplification efficiency graph of 101 primer pairs. Figure 5 The SmartChip real-time PCR system is used to perform fluorescence quantitative PCR on the prepared chip (including 101 pairs of primers). After the PCR is completed, the software will automatically analyze and export the data. The exported data includes the efficiency data, and then the efficiency data is plotted. Figure 5 . Amplification efficiency calculation formula: Eff = 10 (-1 / slope) -1, where slope is the slope corresponding to the standard curve.

[0052] 7. Results Analysis

[0053] The absolute abundance of resistance genes in sewage samples from five parts of the sewage treatment plant, including the high-concentration antibiotic inlet section (INF-H), low-concentration antibiotic inlet section (INF-L), aerobic tank (OT), anaerobic tank (AT) and effluent section (EFF), is the average value of three parallel samples. The results are shown in Table 2.

[0054] Table 2 Absolute abundance of resistance genes in sewage treatment plants

[0055]

[0056]

[0057]

[0058]

[0059] Note: total is the sum of the abundance of these 101 genes.

[0060] As can be seen from Table 2, resistance genes of different abundances were detected in the inlet section, aerobic tank, anaerobic tank and effluent section of the sewage treatment plant. Among them, the absolute abundance of resistance genes in the effluent section was significantly lower than that in the inlet section, indicating that the resistance genes detected in the effluent section were significantly less than those in the inlet section. Consistent with the actual situation, the detection method is accurate. It can be seen that the high-throughput absolute quantitative detection primers, chips and detection methods for microbial resistance genes can accurately and efficiently characterize the exposure of resistance genes in the environment. Therefore, the present invention has broad application prospects for quantifying and evaluating the risks of resistance genes in complex environmental sample systems.

[0061] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A high-throughput detection primer set for microbial antibiotic resistance genes, characterized in that: The primer set contains 101 pairs of primer sequences, and the sequences of the 101 pairs of primers are shown as SEQ ID NOs: 1-202.

2. A high-throughput quantitative detection chip for microbial resistance genes, characterized in that: Contains the primer set according to claim 1.

3. The high-throughput quantitative detection chip according to claim 2, characterized in that: It also contains a positive clone plasmid; the positive clone plasmid is a plasmid with a resistance gene inserted, and the resistance gene is any gene amplified by the primer set according to claim 1.

4. A high-throughput quantitative detection kit for microbial resistance genes, characterized in that: Contains the primer set according to claim 1.

5. The high-throughput quantitative detection kit according to claim 4, characterized in that: It also contains a positive clone plasmid; the positive clone plasmid is a plasmid with a resistance gene inserted, and the resistance gene is any gene amplified by the primer set according to claim 1.

6. Use of the primer set of claim 1, the detection chip of any one of claims 2-3, or the detection kit of any one of claims 4-5 for detecting microbial resistance genes in environmental samples.

7. A high-throughput quantitative detection method for microbial resistance genes for non-diagnostic purposes, characterized in that: The following steps are included: Extracting total DNA from samples to be tested; Amplification: using the primer set of claim 1 or the detection chip of any one of claims 2-3 or the detection kit of any one of claims 4-5 to perform PCR amplification on the sample to be tested; Result analysis: The abundance of microbial resistance genes was absolutely quantified using the standard plasmid external standard method.

8. The detection method according to claim 7, characterized in that: The PCR amplification procedure was: pre-denaturation at 95°C for 10 min; followed by denaturation at 95°C for 30 s, annealing and extension at 60°C for 30 s, and 40 cycles.

Images