A degenerate primer pair for detecting the tet(X) gene and its homologous genes and a method thereof

Abstract

The present invention belongs to the field of molecular biology, and specifically discloses a degenerate primer pair for detecting the tet(X) gene and its homologous genes and a method therefor. The degenerate primers are designed based on the multiple sequence alignment of the tet(X) gene and its homologous genes, and have good specificity and sensitivity. By optimizing the PCR parameters, a PCR method for rapidly detecting the tet(X) gene and its homologous genes is established. Repeated experiments have proven that this method is rapid, sensitive, and can detect multiple tet(X) homologous genes with a pair of primers, with low detection cost, short time consumption, and good universality. It provides an efficient and reliable method for the epidemiological investigation of drug-resistant genes.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to a degenerate primer pair for detecting the tet(X) gene and its homologous genes and a method therefor. Background Art

[0002] Tigecycline is a third-generation tetracycline antibiotic and is considered the "last line of defense" for clinical treatment against multidrug-resistant bacteria. In recent years, the tigecycline resistance gene tet(X) and its homologous genes have been widely present in bacteria from various sources such as the livestock and poultry breeding industry, livestock and poultry products, humans, and the environment. This gene can confer high-level resistance of bacteria to tetracycline antibiotics, seriously threatening human health. Monitoring the prevalence of the tet(X) gene and its homologous genes helps to guide rational clinical drug use and timely control the spread of drug-resistant bacteria and drug-resistant genes.

[0003] tet(X) was initially discovered on the R plasmid of the obligate anaerobic Bacteroides fragilis and encodes a flavin-dependent monooxygenase. Subsequently, the homologous genes tet(X1) and tet(X2) were also discovered in Bacteroides, which have 61.7% and 99.5% amino acid sequence identity with tet(X) respectively, but no large-scale prevalence was found. Until 2019, it was found that the novel tet(X) homologs tet(X3) and tet(X4) genes were widely present in animal-derived Escherichia coli and Acinetobacter, which attracted great attention from global scholars. Subsequently, various variants were found in Flavobacterium, Sphingobacterium, Myroides, and multidrug-resistant pathogenic bacteria (such as Klebsiella pneumoniae, Enterobacter, Pseudomonas, etc.). In particular, Riemerella anatipestifer in the family Flavobacteriaceae has now been found to have 44 tet(X) homologous genes, with homology ranging from 60% to 99%.

[0004] Currently, the methods for detecting the tet(X) gene and its homologous genes mainly rely on PCR amplification and Sanger sequencing. However, different primers need to be selected for different tet(X) homologous genes, and multiple amplifications are required to complete the detection. There is also a method using multiplex PCR for detection, but the detection effect is unstable, and many uncommon homologous genes cannot be detected, resulting in missed detections. It is only applicable to the common tet(X3) and tet(X4) genes, with great limitations and being relatively cumbersome, which is not conducive to the epidemiological investigation of drug-resistant genes.

[0005] As disclosed in Chinese Patent Application No. 201911069274.5, a fluorescence quantitative PCR primer composition for detecting tigecycline resistance gene tet(X) and its variants and its application are provided. Multiple sets of primers are designed for five variants of tigecycline resistance gene tet(X), namely tet(X), tet(X1), tet(X2), tet(X3), and tet(X4), to establish corresponding fluorescence quantitative PCR methods. This detection method is applicable to culturable and non-culturable bacteria, and the PCR reaction conditions are the same, thus ensuring that five variants of tigecycline resistance gene tet(X) in the sample can be detected simultaneously under the same PCR conditions. However, different primers need to be selected for different tet(X) homologous genes, and multiple amplifications are required to complete the detection.

[0006] Chinese Patent Application No. 202010862590.4 discloses a method for rapidly detecting the presence of tet(X) gene in strains. Based on the function that the enzyme expressed by the tet(X) gene can degrade tigecycline, a method using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is developed, which can only detect whether the strain carries the tet(X) gene, but cannot detect tet(X) homologous genes.

[0007] Therefore, there is an urgent need to develop a degenerate primer pair and its method for detecting the tet(X) gene and its homologous genes. Summary of the Invention

[0008] To solve the above technical problems, the present invention provides a degenerate primer pair and its method for detecting the tet(X) gene and its homologous genes, which can achieve the detection of multiple tet(X) homologous genes with a pair of primers.

[0009] To achieve the above object, the present invention is implemented according to the following technical scheme:

[0010] The first object of the present invention is to provide a degenerate primer pair for detecting the tet(X) gene and its homologous genes, and the degenerate primer pair includes an upstream primer and a downstream primer;

[0011] The nucleotide sequence of the upstream primer is: 5’-GGHGTAAAYWTTGYTGATGA-3’;

[0012] The nucleotide sequence of the downstream primer is: 5’-GGTTSADGAATRTCRSCYTG-3’;

[0013] Wherein: W = A or T; Y = C or T; S = C or G; R = A or G; D = A, G or T.

[0014] The second object of the present invention is to provide a method for detecting the tet(X) gene and its homologous genes, comprising the following steps:

[0015] S1. Extract the DNA of the strain to be tested;

[0016] S2. Using the extracted DNA as a template, construct a PCR reaction system and / or a qPCR reaction system with the above-mentioned degenerate primer pairs for detecting the tet(X) gene and its homologous genes, and perform PCR reaction or qPCR reaction;

[0017] S3. Perform gel electrophoresis separation and staining on the PCR products, and judge the results: if a 332bp band appears, it indicates that the strain to be tested carries the tet(X) gene or its homologous gene; otherwise, it indicates that the strain to be tested does not carry the tet(X) gene or its homologous gene; analyze the Ct value of the qPCR products, if the Ct value is less than 30, it indicates that the strain to be tested carries the tet(X) gene or its homologous gene, otherwise, it indicates that the strain to be tested does not carry the tet(X) gene or its homologous gene;

[0018] S4. Perform PCR reaction on the DNA of the strain to be tested, and perform first-generation sequencing on the PCR products, and compare the sequences with the tet(X) gene or its homologous genes in the Genbank database to determine the specific tet(X) variant.

[0019] Specifically, in the step S2, the PCR reaction system is as follows: taking 20 μL as an example, 10 μL of 2×Taq Master Mix (Hangzhou Qingke Biotechnology Co., Ltd.), 0.5 μL of 10 mmol / L upstream primer, 0.5 μL of 10 mmol / L downstream primer, 1 μL of 200 μg / μL template DNA, and 8 μL of ddH2O;

[0020] The PCR reaction conditions are: 1) Pre-denaturation at 95°C for 5 min; 2) Denaturation at 95°C for 30 s, annealing at 42 - 52°C for 45 s; extension at 72°C for 20 - 60 s, for a total of 30 - 40 cycles; 3) Extension at 72°C for 5 min.

[0021] Specifically, in the step S2, the qPCR reaction system is as follows: taking 20 μL as an example, 10 μL of 2×TB Green Fast qPCRMix, 0.5 μL of 10 mmol / L upstream primer, 0.5 μL of 10 mmol / L downstream primer, 1 μL of 200 μg / μL template DNA, and 8 μL of ddH2O;

[0022] The qPCR reaction conditions are: 1) Pre-denaturation at 95°C for 2 min; 2) Denaturation at 95°C for 30 s, annealing at 48°C for 30 s, extension at 72°C for 20 s, for a total of 40 cycles; 3) Extension at 72°C for 5 min.

[0023] Preferably, the PCR reaction conditions are as follows: 1) pre-denaturation at 95°C for 5 min; 2) denaturation at 95°C for 30 s, annealing at 48°C for 45 s, extension at 72°C for 45 s, for a total of 35 cycles; 3) extension at 72°C for 5 min.

[0024] Compared with the prior art, the primer pair of the present invention is designed based on the multiple sequence alignment of the tet(X) gene and its homologous genes, and has good specificity and sensitivity; by optimizing the PCR parameters, a PCR method for rapidly detecting the tet(X) gene and its homologous genes is established. Repeated experiments have proved that this method is rapid, sensitive, and can detect multiple tet(X) homologous genes with a pair of primers, with low detection cost, short time consumption, and good universality. It provides an efficient and reliable method for the epidemiological investigation of drug-resistant genes. Description of the Drawings

[0025] Figure 1 Multiple sequence alignment of the tet(X) gene and its homologous genes.

[0026] Figure 2 To screen the optimal temperature for PCR amplification.

[0027] Figure 3 qPCR amplification curve diagram for detecting the strain to be tested using the primer.

[0028] Figure 4 Gel electrophoresis diagram after PCR amplification of the strain to be tested using the primer. Detailed Embodiments

[0029] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0030] Unless otherwise specified, the methods in the following embodiments are all conventional methods in the art. The strains used in the following embodiments are shown in Table 1, and all the strains are preserved in the laboratory.

[0031]

[0032]

[0033] Example 1

[0034] In order to design a degenerate primer pair capable of detecting the tet(X) gene and its homologous genes, the tet(X) gene and its homologous genes in the Genebank database were collected for sequence alignment (as Figure 1As shown, several relatively conserved regions were found, with lengths reaching over 20 bp and only individual base mutations. Based on this discovery, the present invention covers all mutant bases by designing degenerate primers, replacing these mutant sites with degenerate bases, such as Figure 1 As shown; synthesize primer pairs capable of amplifying the tet(X) gene and all known tet(X) homologous genes, and it can be determined whether the test strain carries the tet(X) gene and its homologous genes according to whether the target product can be amplified.

[0035] The degenerate primer pairs for detecting the tet(X) gene and its homologous genes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The degenerate primer pairs include upstream primers and downstream primers;

[0036] The nucleotide sequence of the upstream primer is: 5'-GGHGTAAAYWTTGYTGATGA-3';

[0037] The nucleotide sequence of the downstream primer is: 5'-GGTTSADGAATRTCRSCYTG-3';

[0038] Where: W = A or T; Y = C or T; S = C or G; R = A or G; D = A, G or T.

[0039] Example 2

[0040] The tet(X) gene and its homologous genes can be detected by performing PCR amplification using the degenerate primer pairs for detecting the tet(X) gene and its homologous genes in Example 1. The specific operations are as follows:

[0041] Using the DNA of Escherichia coli without the tet(X) homologous gene as a blank control and the DNA of Escherichia coli known to carry the tet(X) homologous gene as a positive control. Isolate and culture strains carrying different tet(X) homologous genes, pick single colonies and amplify them in broth medium, and then extract the bacterial genomic DNA in Table 1 using a bacterial DNA extraction kit (Shanghai Jierui Biotech Co., Ltd.).

[0042] Using the extracted DNA as a template, and using the primers of Example 1, amplification was carried out by PCR method; the PCR reaction system was as follows: calculated by 20 μL, 2×Taq Master Mix (Hangzhou Qingke Biotechnology Co., Ltd.) 10 μL, 10 mmol / L upstream primer 0.5 μL, 10 mmol / L downstream primer 0.5 μL, 200 μg / μL template DNA 1 μL, ddH2O 8 μL; the PCR reaction conditions were: 1) pre-denaturation at 95 °C for 5 min; 2) denaturation at 95 °C for 30 s, annealing at 42 - 52 °C for 45 s; extension at 72 °C for 20 - 60 s, for a total of 30 - 40 cycles; 3) extension at 72 °C for 5 min. The annealing temperature of PCR was optimized by gradient PCR method, and the obtained optimal annealing temperature was 48 °C, and the specific gel diagram was as shown in Figure 2 shown. Therefore, the subsequent PCR reaction conditions were preferably: 1) pre-denaturation at 95 °C for 5 min; 2) denaturation at 95 °C for 30 s, annealing at 48 °C for 45 s, extension at 72 °C for 45 s, for a total of 35 cycles; 3) extension at 72 °C for 5 min.

[0043] On this basis, Acinetobacter seohaensis carrying the tet(X3) gene, Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae carrying the tet(X4) gene, Proteus cibarius carrying the tet(X6) gene, and Riemerella anatipestifer carrying different tet(X) homologous genes were used as detection objects, the positive control was Escherichia coli carrying tet(X4), and the negative control was Escherichia coli not carrying tet(X4).

[0044] Single colonies were picked and amplified in broth medium, and then genomic DNA was extracted using a bacterial DNA extraction kit (Shanghai Jierui Bio-Engineering Co., Ltd.).

[0045] Using the degenerate primer pair for detecting the tet(X) gene and its homologous genes in Example 1 to construct a PCR reaction system: calculated by 20 μL, 2×Taq Master Mix (Hangzhou Qingke Biotechnology Co., Ltd.) 10 μL, 10 mmol / L upstream primer 0.5 μL, 10 mmol / L downstream primer 0.5 μL, 200 μg / μL template DNA 1 μL, ddH2O 8 μL; the PCR reaction conditions were preferably: 1) pre-denaturation at 95 °C for 5 min; 2) denaturation at 95 °C for 30 s, annealing at 48 °C for 45 s, extension at 72 °C for 45 s, for a total of 35 cycles; 3) extension at 72 °C for 5 min;

[0046] The PCR products were separated by gel electrophoresis (120 V, 30 min), stained, and the product bands were observed. The results were as Figure 4 shown. Bands of 322 bp could be amplified from A. seohaensis, E. coli, K. pneumoniae, E. cloacae, P. cibarius, and R. anatipestifer carrying the tet(X) homologous gene, while no 322-bp band could be amplified from E. coli without the tet(X) homologous gene.

[0047] Then, the PCR products were sequenced by first-generation sequencing, and the sequences were aligned with the tet(X3), tet(X4), and tet(X6) genes in the Genbank database. When the sequences were the same, the specific homologous gene could be determined.

[0048] Example 3

[0049] Acinetobacter seohaensis carrying the tet(X3) gene, Escherichia coli carrying the tet(X4) gene, Klebsiella pneumoniae, Proteus cibarius carrying the tet(X6) gene, and Riemerella anatipestifer carrying different tet(X) homologous genes were used as the detection objects.

[0050] Single colonies were picked and amplified in broth medium, and then genomic DNA was extracted using a bacterial DNA extraction kit (Shanghai Jierui Bio-engineering Co., Ltd.).

[0051] A qPCR reaction system (20 μL) was constructed using the degenerate primers for detecting the tet(X) gene and its homologous genes in Example 1: 10 μL of 2×TB Green Fast qPCR Mix, 1.5 μL of 10 mmol / L upstream primer, 1.5 μL of 10 mmol / L downstream primer, 1 μL of 200 μg / μL template DNA, and 6 μL of ddH2O; qPCR reaction conditions: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 48°C for 30 s, extension at 72°C for 20 s, for a total of 50 cycles; finally, extension at 72°C for 2 min. The instrument used for fluorescence quantitative PCR was Bio-Rad (CFX96). Fluorescence quantitative PCR was performed on the DNA of the cultured colonies, and the obtained fluorescence quantitative amplification curves were as Figure 3 shown, and the specific Ct values are shown in Table 2.

[0052] The Ct value of the known tet(X4) gene positive control is below 35, and the Ct values of the tested strains are all below 35. This indicates that these strains carry the tet(X) homologous genes. The Ct value of the negative control without the tet(X) homologous gene is above 35.

[0053] Table 2

[0054]

[0055]

[0056] Perform PCR reaction on the DNA of the tested strains (the same as in Example 2), and perform first-generation sequencing on the PCR products. Compare the sequences with the tet(X3) gene, tet(X4) gene, and tet(X6) gene in the Genbank database. When the sequences are the same, the specific homologous gene can be determined.

[0057] The above examples only exemplarily detect the tet(X3) gene, tet(X4) gene, and tet(X6) gene. The above method can also be used to detect the tet(X) gene and its homologous genes tet(X1)-tet(X14), which will not be elaborated in this example.

[0058] In summary, the present invention has established a PCR method for rapid detection of tet(X) homologous genes. Repeated experiments have proven that this method is rapid, sensitive, and can detect the tet(X) gene and its homologous genes tet(X1)-tet(X14) with a pair of primers, with low detection cost, short time consumption, and good universality.

[0059] The technical solution of the present invention is not limited to the limitations of the above specific embodiments. Any technical deformation made according to the technical solution of the present invention falls within the protection scope of the present invention.

Claims

1. A detection tet of degenerate primer pairs for (X) gene and its homologous genes, characterized in that: The degenerate primer pair includes an upstream primer and a downstream primer; The nucleotide sequence of the upstream primer is: 5’-GGHGTAAAYWTTGYTGATGA-3’; The nucleotide sequence of the downstream primer is: 5’-GGTTSADGAATRTCRSCYTG-3’; Where: H = A, C or T, W = A or T; Y = C or T; S = C or G; R = A or G; D = A, G or T.

2. A method for detecting tet (X) gene and its homologous genes, characterized in that It includes the following steps: S1. Extract the DNA of the strain to be tested; S2. Using the extracted DNA as a template, construct a PCR reaction system and / or a qPCR reaction system with the degenerate primer pair of the (X) gene and its homologous genes, and perform a PCR reaction or a qPCR reaction; tet (X) gene and its homologous genes of the degenerate primer pair to construct a PCR reaction system and / or qPCR reaction system, for PCR reaction or qPCR reaction; S3. Gel electrophoresis separation, staining, and result judgment of the PCR product: If a 332-bp band appears, it indicates that the tested strain carries tet (X) gene or its homologous gene; Otherwise, it indicates that the strain to be tested has no tet (X) gene or its homologous gene; Analyze the Ct value of the qPCR product. If the Ct value is less than 35, it indicates that the strain to be tested carries tet (X) gene or its homologous gene. Otherwise, it indicates that the strain to be tested does not have tet (X) gene or its homologous gene; S4. Perform a PCR reaction on the DNA of the strain to be tested, and perform first-generation sequencing on the PCR product. Compare the sequence with the tet (X) gene or its homologous gene to determine the specific tet (X) variant.

3. The detection according to claim 2 tet (X) gene and its homologous genes, characterized in that In the step S2, the PCR reaction system is as follows (taking 20 μL as an example): 2×Taq Master Mix 10 μL, 10 mmol / L upstream primer 0.5 μL, 10 mmol / L downstream primer 0.5 μL, 200 μg / μL template DNA 1 μL, ddH2O 8 μL; The PCR reaction conditions are: 1) Pre-denaturation at 95°C for 5 min; 2) Denaturation at 95°C for 30 s, annealing at 42 - 52°C for 45 s; extension at 72°C for 20 - 60 s, for a total of 30 - 40 cycles; 3) Extension at 72°C for 5 min.

4. The detection according to claim 2 tet (X) gene and its homologous genes, characterized in that In the step S2, the qPCR reaction system is as follows (taking 20 μL as an example): 2×TB Green Fast qPCR Mix 10 μL, 10 mmol / L upstream primer 1.5 μL, 10 mmol / L downstream primer 1.5 μL, 200 μg / μL template DNA 1 μL, ddH2O 6 μL; The qPCR reaction conditions are: 1) Pre-denaturation at 95°C for 3 min; 2) Denaturation at 95°C for 30 s, annealing at 48°C for 30 s, extension at 72°C for 20 s, for a total of 40 cycles; 3) Extension at 72°C for 2 min.

5. The detection according to claim 3 tet (X) gene and its homologous gene, characterized in that The PCR reaction conditions are: 1) Pre-denaturation at 95°C for 5 min; 2) Denaturation at 95°C for 30 s, annealing at 48°C for 45 s, extension at 72°C for 45 s, for a total of 35 cycles; 3) Extension at 72°C for 5 min.

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