Primer set, kit and application thereof for high-throughput targeted detection of ruminant pathogens and drug-resistant genes

Abstract

The present invention discloses a primer set, a kit and an application thereof for high-throughput targeted detection of ruminant pathogens and drug-resistant genes. The primer set is composed of 140 pairs of primers with nucleotide sequences such as SEQ ID NO: 102-381, and is used to amplify pathogen target genes and drug-resistant genes. The primer set, the kit and the detection method of the present invention sequence specific regions of 83 pathogen target genes and 18 drug-resistant genes of ruminants, thereby improving the accuracy and specificity of the detection, being able to detect multiple related pathogens in one experiment, being able to detect trace amounts of pathogenic nucleic acids, and being helpful for early diagnosis; being able to complete the detection of a large number of samples in a short time, improving the detection efficiency, and having a lower cost than whole genome sequencing; being only targeted at specific regions, the complexity of data analysis is reduced, and being applicable to various clinical samples, such as blood, tissues, secretions, etc., providing a unified and effective method for detecting different types of samples.

Description

Technical Field

[0001] The present invention relates to the technical field of pathogenic organism detection, and more specifically, to a primer set, a kit and applications thereof for high-throughput targeted detection of ruminant pathogens and drug-resistant genes. Background Art

[0002] Bovine mastitis (BM), bovine respiratory disease (BRD), and bovine diarrhea Bovine mastitis (BED) is an important disease in ruminants and beef cattle. There are many types of pathogenic microorganisms that cause ruminant mastitis, including bacteria, fungi, mycoplasma, viruses, etc. At present, more than 150 types of ruminant mastitis pathogens have been reported, which are often caused by the coexistence of dominant bacteria and several pathogenic microorganisms, rather than a single pathogen. Bacterial pathogens are one of the main causes of bovine respiratory diseases, and it is often common to have mixed infections with multiple pathogens and secondary infections with other pathogens. Viral pathogens are also one of the important factors leading to the occurrence of respiratory diseases. Common viral pathogens include parainfluenza type 3 virus (BPIV3) and bovine viral diarrhea virus (BVDV). Bovine digestive tract diseases are often manifested as mixed infections of multiple pathogens such as bacteria, viruses, and parasites. Common pathogens include Escherichia coli, Salmonella enteritidis, bovine viral diarrhea virus, bovine enterovirus, etc.

[0003] At present, the main methods for detecting pathogens include bacterial isolation and identification, serological detection, gene chips, PCR detection technology and gene sequencing technology. Based on the genomic region being detected, gene sequencing technology involves three basic sequencing types: Metagenome sequencing (mNGS), whole exome sequencing (WES) and targeted Next Generation Sequencing (tNGS). The existing detection methods for ruminant pathogens have problems such as low sensitivity, poor specificity, incomplete target coverage, long process time, low detection efficiency and high detection cost, and are not suitable for the initial screening of ruminant pathogens. In order to solve the above problems, it is necessary to develop high-throughput targeted detection primers, kits and corresponding detection methods for ruminant pathogens and drug-resistant genes. Summary of the invention

[0004] The purpose of the present invention is to provide a primer set, a kit and applications thereof for high-throughput targeted detection of ruminant pathogens and drug-resistant genes.

[0005] According to one aspect of the present invention, a primer set for high-throughput targeted detection of ruminant pathogens and drug-resistant genes is provided, the primer set consisting of 140 pairs of primers with nucleotide sequences as shown in SEQ ID NOs: 102 to 381, each pair of primers consisting of an upstream primer and a downstream primer, and the 140 pairs of primers are used to amplify pathogen target genes and drug-resistant genes.

[0006] In some embodiments, the 140 pairs of primers are respectively directed to 83 pathogenic target genes and 18 drug-resistant genes of ruminants, each pathogenic target gene or drug-resistant gene is provided with 1 to 2 pairs of primers, and the sequences of the 83 pathogenic target genes and 18 drug-resistant genes are shown in SEQ ID NOs: 1 to 101.

[0007] In some embodiments, the even-numbered sequences in each pair of primers are upstream primers, the odd-numbered sequences in each pair of primers are downstream primers, and both the upstream primers and the downstream primers carry a linker sequence.

[0008] In some embodiments, the linker sequence of the upstream primer is ATCACGAC, and the linker sequence of the downstream primer is TGAACCTT.

[0009] According to another aspect of the present invention, there is provided the application of the above-mentioned primer set for high-throughput targeted detection of ruminant pathogens and drug-resistant genes for detecting or assisting in the detection of 83 pathogenic target genes and 18 drug-resistant genes of ruminants in non-disease diagnosis destinations.

[0010] According to another aspect of the present invention, a kit for high-throughput targeted detection of ruminant pathogens and drug-resistant genes is provided, and the kit comprises the above-mentioned primer set for high-throughput targeted detection of ruminant pathogens and drug-resistant genes.

[0011] In some embodiments, the kit further comprises a sample total DNA / RNA extraction reagent, a reverse transcription reagent, and a PCR amplification reagent.

[0012] According to a fourth aspect of the present invention, there is provided the use of the above-mentioned high-throughput targeted detection kit for ruminant pathogens and drug-resistant genes in detecting 83 pathogen target genes and 18 drug-resistant genes in ruminants, wherein the ruminants are cattle.

[0013] According to a fifth aspect of the present invention, a method for high-throughput targeted detection of ruminant pathogens and drug-resistant genes is provided, the method using the above-mentioned high-throughput targeted detection kit for ruminant pathogens and drug-resistant genes, the method is a method for non-disease diagnosis purposes, and comprises the following steps:

[0014] S1. Total DNA / RNA nucleic acid co-extraction of the sample to be tested;

[0015] S2. Reverse transcription;

[0016] S3. Use the above-mentioned high-throughput targeted detection kit for ruminant pathogens and drug-resistant genes to perform multiple targeted PCR amplifications in sequence, add adapter amplification, and obtain a sequencing library;

[0017] S4. After quality inspection of the sequencing library, high-throughput sequencing is performed on the machine to obtain sequencing data, which is then analyzed to obtain the detection results of pathogen target genes and drug-resistant genes.

[0018] In some embodiments, the concentration of DNA / RNA extracted in step S1 is ≥ 0.1 ng / uL.

[0019] The beneficial effects of the present invention are as follows: the primer set, the kit and the detection method of the present invention sequence specific regions of 83 pathogen target genes and 18 drug-resistant genes of ruminants, thereby improving the accuracy and specificity of the detection, being able to detect a variety of related pathogens in one experiment, being able to detect trace amounts of pathogenic nucleic acids, and being helpful for early diagnosis; a large number of samples can be tested in a short time, thereby improving the detection efficiency, and compared with whole genome sequencing, the targeted sequencing has a lower cost; since only specific regions are targeted, the complexity of data analysis is reduced, and the method is applicable to various clinical samples, such as blood, tissues, secretions, etc., providing a unified and effective method for testing different types of samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a flow chart of the detection method of Example 3 of the present invention.

[0021] Figure 2 This is a diagram showing the gene sequencing identification results of SEQ ID NOs: 1 to 55 in Example 4 of the present invention.

[0022] Figure 3 This is a diagram showing the gene sequencing identification results of SEQ ID NOs: 56 to 83 in Example 4 of the present invention.

[0023] Figure 4 This is a diagram showing the sequencing and identification results of the drug-resistant genes of SEQ ID NOs: 84 to 101 in Example 4 of the present invention. DETAILED DESCRIPTION

[0024] The present invention will be described clearly and completely below in conjunction with the embodiments and drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments that can be obtained by those of ordinary skill in the art based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0025] Example 1 Primer set for high-throughput targeted detection of ruminant pathogens and drug-resistant genes

[0026] Common pathogens in ruminant mastitis, respiratory tract, and digestive tract were selected through statistics and analysis, and the pathogen target genes were determined. The complete gene sequence was downloaded from NCBI (https: / / www.ncbi.nlm.nih.gov / ) according to the target gene name, and the specificity of the target gene was further determined by NCBI Blast, and finally the sequence of the pathogen target gene was obtained. The sequences of the pathogen target genes and drug resistance genes targeted by the primer set for high-throughput targeted detection of ruminant pathogens and drug resistance genes of the present invention are shown in SEQ ID NO.1 to SEQ ID NO.101. See Table 1 for details.

[0027] Table 1 List of 83 pathogen target genes and 18 drug resistance genes in ruminants

[0028]

[0029]

[0030]

[0031]

[0032] According to the above pathogen target gene and drug resistance gene sequences (SEQ ID NO.1 to SEQ ID NO.101), multiple designs, analyses and comparisons were performed, and 1 to 2 pairs of specific primers were designed for each gene, totaling 140 pairs of primers. The above 140 pairs of primers were synthesized by Shanghai Lingen Biotechnology Co., Ltd., and their nucleotide sequences are shown in SEQ ID NO.102 to SEQ ID NO.382. See Table 2 for details.

[0033] Table 2 Primer sequence list for high-throughput targeted detection of ruminant pathogens and drug resistance genes

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042] Example 2 Kit for high-throughput targeted detection of ruminant pathogens and drug-resistant genes

[0043] The components of the kit for high-throughput targeted detection of ruminant pathogens and drug-resistant genes of this embodiment include the primer set obtained in Example 1 (sequences are shown in SEQ ID NO.102 to SEQ ID NO.381), DNA / RNA extraction reagents, reverse transcription reagents, and PCR amplification reagents. The main contents of each component are shown in Table 3.

[0044] Table 3 Components of the kit for high-throughput targeted detection of ruminant pathogens and drug-resistant genes

[0045]

[0046] Note: Except for the primer set, all reagent components in Table 3 are commercially available conventional reagents.

[0047] Example 3 A method for high-throughput targeted detection of ruminant pathogens and drug-resistant genes

[0048] The primer set of Example 1 and the kit of Example 2 were used to construct the library. Figure 1 The specific steps are as follows:

[0049] S1. Use DNA / RNA extraction reagent to extract total DNA / RNA nucleic acid from the sample to be tested, and measure the concentration of the extracted nucleic acid;

[0050] S2. Reverse transcribe the RNA by adding reverse transcription buffer and treating at 42°C for 2 min, then adding reverse transcriptase and treating at 37°C for 15 min, and then treating at 85°C for 5 s;

[0051] S3. Use the kit for high-throughput targeted detection of ruminant pathogens and drug-resistant genes in Example 2 to perform multiple targeted PCR amplifications in sequence, and use 1 μg of DNA as the starting amount to build a library. Use Covaris M220 ultrasound to shear the DNA to about 450 bp, fill in the blank, add A at the 3' end, connect the index adapter (TruSeq™ Nano DNA Sample Prep Kit), enrich the library, perform PCR amplification for 8 cycles, obtain a sequencing library, and use 2% agarose gel to recover the target band (Certified Low Range Ultra Agarose) to recover the library;

[0052] S4. Perform quality inspection on the sequencing library, quantify it with TBS380 (Picogreen), mix it according to the data ratio, perform bridge PCR amplification on cBot to generate clusters, perform 2*150bp sequencing on the Illumina Hiseq sequencing platform to obtain sequencing data, and then analyze the sequencing data to obtain the detection results of pathogen target genes and drug-resistant genes.

[0053] Example 4 Method Performance Evaluation

[0054] The genomes or plasmids of 83 pathogenic target genes and 18 drug-resistant genes of ruminants shown in Table 1 of Example 1 were diluted, and the templates were mixed according to the ratio of 104 copies of each detection target to prepare sample 1, sample 2 and sample 3 simulating clinical samples. The primer set of Example 1, the kit of Example 2 and the detection method of Example 3 were used to construct tNGS libraries and perform sequencing analysis. The detection results are shown in Table 4 and Figure 2~Figure 4 .

[0055] Table 4 Detection results of common pathogen target genes and drug resistance genes in ruminants

[0056]

[0057] As can be seen from Table 4, the primer set, kit and detection method of the present invention can be used to detect mastitis pathogens, respiratory tract pathogens and digestive tract pathogens in cattle, and the detection results are accurate and reliable.

[0058] The primer set, kit and detection method of the present invention sequence specific pathogenic gene regions of ruminants, thereby improving the accuracy and specificity of detection, being able to detect multiple related pathogens in one experiment, and being able to detect trace amounts of pathogenic nucleic acids, which is helpful for early diagnosis; a large number of samples can be tested in a short time, thereby improving the detection efficiency, and the cost of targeted sequencing is more controllable compared to whole genome sequencing; since only specific regions are targeted, the complexity of data analysis is reduced, and the method is applicable to various clinical samples, such as blood, tissues, secretions, etc., providing a unified and effective method for testing different types of samples.

[0059] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements may be made without departing from the inventive concept of the present invention, which all fall within the protection scope of the present invention.

Claims

1. A primer set for high-throughput targeted detection of ruminant pathogens and drug-resistant genes, characterized in that: The primer set consists of 140 pairs of primers with nucleotide sequences as shown in SEQ ID NOs: 102-381, each pair of primers consists of an upstream primer and a downstream primer, and the 140 pairs of primers are used to amplify pathogen target genes and drug resistance genes.

2. The primer set for high-throughput targeted detection of ruminant pathogens and drug-resistant genes according to claim 1, characterized in that: The 140 pairs of primers are respectively directed to 83 pathogenic target genes and 18 drug-resistant genes of ruminants, each pathogenic target gene or drug-resistant gene is provided with 1-2 pairs of primers, and the sequences of the 83 pathogenic target genes and the 18 drug-resistant genes are shown in SEQ ID NOs: 1-101.

3. The primer set for high-throughput targeted detection of ruminant pathogens and drug-resistant genes according to claim 1, characterized in that: The even-numbered sequences in each pair of primers are upstream primers, and the odd-numbered sequences in each pair of primers are downstream primers. Both the upstream primers and the downstream primers carry a linker sequence.

4. The primer set for high-throughput targeted detection of ruminant pathogens and drug-resistant genes according to claim 3, characterized in that: The linker sequence of the upstream primer is ATCACGAC, and the linker sequence of the downstream primer is TGAACCTT.

5. The use of the primer set for high-throughput targeted detection of ruminant pathogens and drug-resistant genes according to any one of claims 1 to 4 for detecting or assisting in the detection of 83 pathogenic target genes and 18 drug-resistant genes in ruminants for non-disease diagnosis purposes, wherein the sequences of the 83 pathogenic target genes and the 18 drug-resistant genes are shown in SEQ ID NOs: 1 to 101.

6. A kit for high-throughput targeted detection of ruminant pathogens and drug-resistant genes, characterized in that: The kit comprises the primer set for high-throughput targeted detection of ruminant pathogens and drug-resistant genes according to any one of claims 1 to 4.

7. The kit for high-throughput targeted detection of ruminant pathogens and drug-resistant genes according to claim 6, characterized in that: The kit also includes a sample total DNA / RNA extraction reagent, a reverse transcription reagent, and a PCR amplification reagent.

8. Use of the kit for high-throughput targeted detection of ruminant pathogens and drug-resistant genes according to any one of claims 6-7 for detecting 83 pathogen target genes and 18 drug-resistant genes in ruminants for non-disease diagnosis purposes, wherein the sequences of the 83 pathogen target genes and the 18 drug-resistant genes are shown in SEQ ID NOs: 1 to 101, and the ruminant is a cattle.

9. A method for high-throughput targeted detection of ruminant pathogens and drug-resistant genes, characterized in that: The method uses the kit for high-throughput targeted detection of ruminant pathogens and drug-resistant genes according to any one of claims 6 to 7. The method is a method for non-disease diagnosis purposes and comprises the following steps: S1. Total DNA / RNA nucleic acid co-extraction of the sample to be tested; S2. Reverse transcription; S3. Use the kit for high-throughput targeted detection of ruminant pathogens and drug-resistant genes according to any one of claims 6 to 7 to sequentially perform multiple targeted PCR amplification and adapter amplification to obtain a sequencing library; S4. After quality inspection of the sequencing library, high-throughput sequencing is performed on the machine to obtain sequencing data, which is then analyzed to obtain the detection results of pathogen target genes and drug-resistant genes.

10. A method for high-throughput targeted detection of ruminant pathogens and drug-resistant genes according to claim 9, characterized in that: The concentration of DNA / RNA extracted in step S1 is ≥ 0.1 ng / uL.

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