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Multiplex-PCR (Polymerase Chain Reaction) detection primer group and kit for various duck-derived pathogenic bacteria

A detection kit and pathogenic bacteria technology, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, and resistance to vector-borne diseases, etc. It can solve the problems of long detection cycle, cumbersome operation, and inability to detect in large quantities.

Active Publication Date: 2014-07-23
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The isolation and identification of bacteria is the gold standard for diagnosis, but this method has a long detection cycle, cumbersome operation, low sensitivity and cannot be detected in large quantities
[0008] "Establishment and application of dual PCR method for differential diagnosis of Riemerella anatipestifer and colibacillosis" researched by Qin Zonghua et al. discloses a double PCR method for rapid and accurate differential diagnosis of Riemerella anatipestifer and colibacillosis. However, this method can only detect bacterial diseases of two kinds of ducks at the same time. In order to save costs and improve detection efficiency, it is necessary to seek a method for simultaneous detection of multiple bacterial diseases

Method used

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  • Multiplex-PCR (Polymerase Chain Reaction) detection primer group and kit for various duck-derived pathogenic bacteria
  • Multiplex-PCR (Polymerase Chain Reaction) detection primer group and kit for various duck-derived pathogenic bacteria
  • Multiplex-PCR (Polymerase Chain Reaction) detection primer group and kit for various duck-derived pathogenic bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Primer Design and Primer Specificity Detection

[0036] This embodiment utilizes the different serotypes of R. anatipestifer to be determined by the applicant and submitted to GenBank wxya The sequence of the gene (GenBank accession numbers), and other bacteria published on GenBank, such as Escherichia coli, Streptococcus, Salmonella, etc. wxya The genes were compared, and the regions with large sequence differences were selected to design primers for the detection of R. anatipestifer. The primer pair was RA rpoB-P1 (SEQ ID NO: 1) and RArpoB-P2 (SEQ ID NO: 2 ).

[0037] This embodiment selects the housekeeping gene of Escherichia coli phoA The gene was used as the target gene for the detection of Escherichia coli in ducks. for different bacteria that have been published phoA The primers were redesigned according to gene sequence alignment, and the primer pair was E. coli phoA-P1 (SEQ ID NO: 3) and E. coli phoA-P2 (SEQ ID NO: 4).

[0038] In this examp...

Embodiment 2

[0050] Example 2 Establishment of Multiplex PCR Detection Kit for Duck-derived Pathogenic Bacteria

[0051] Prepare duck-derived pathogenic bacteria multiplex PCR detection kit according to the following composition:

[0052] Contains 10×PCR reaction buffer (100mM Tris-HCl (pH8.3), 500mM KCl, 15mM MgCl 2 ), DNA polymerase (5units / μL), dNTP (2.5mM), and 4 pairs of primers (10uM) are as follows:

[0053] SEQ ID NO: 1: 5'-TGCCCAAGCGAATGTGGAGC-3';

[0054] SEQ ID NO: 2: 5'-ACCGGAAATCTGGTTTGGCG-3';

[0055] SEQ ID NO: 3: 5'-CGATAAGCCCGCAGTCACCT-3';

[0056] SEQ ID NO: 4: 5'-GACCAGCGTGTTACCCTCCT-3';

[0057] SEQ ID NO: 5: 5'-TACCAGAAAGGGACGGCTAA-3';

[0058] SEQ ID NO: 6: 5'-CGTTTACGGCGGCGTGGACTACC-3';

[0059] SEQ ID NO: 7: 5'-TGGGTAACGCATGAAGAGGG-3';

[0060] SEQ ID NO: 8: 5'-GGGTCAAGGCTGAGGAAGGT-3'.

[0061] The present invention optimizes the above conditions, and finally determines the reaction system of the kit as follows: 10×PCR reaction buffer 5 μL, DNA polymerase 0....

Embodiment 3

[0065] The specificity of the described multiplex PCR detection kit of embodiment 3

[0066] Prepare 107 cfu / mL of Reeserella anatipestifer (serotype 1), Escherichia coli (ATCC8099), Streptococcus (CVCC556), Salmonella typhimurium, Pasteurella avium, Staphylococcus aureus, Pseudomonas aeruginosa and Mycoplasma, etc. 18 strains of bacteria, the DNA templates of 18 strains of bacteria were extracted by boiling method. Single strain DNA, two-two mixed strain DNA, three-three mixed strain DNA and four-strain mixed strain DNA extracted from 4 strains of R. anatipestifer, Escherichia coli, streptococcus and Salmonella were used as templates, Pasteurella avian, golden yellow Single-strain DNA extracted from four strains of Staphylococcus aeruginosa, Pseudomonas aeruginosa and Mycoplasma was used as a template, and a PCR reaction was performed under the optimized PCR conditions described in Example 2 to determine the specificity of the multiplex PCR reaction. The PCR reaction product...

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Abstract

The invention relates to the technical field of biological detection, and particularly discloses a multiplex-PCR (Polymerase Chain Reaction) detection primer group and kit for various duck-derived pathogenic bacteria. The primer group comprises four pairs of primers which are RArpoB-P1 and RArpoB-P2, E.coliphoA-P1 and E.coliphoA-P2, SalminvA-P1 and SalminvA-P2, Strep16srRNA-P1 and Strep16srRNA-P2. The four pairs of primers are used for carrying out multiplex-PCR amplification and can be used for simultaneously, rapidly, conveniently, specifically and sensitively detecting four bacteria which are riemerella anatipestifer, escherichia coli, salmonella and streptococcus. The four pairs of primers can be used for identification of bacteria, disease diagnosis and epidemiological investigation and has broad market prospect and high economic benefits.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a multiplex PCR detection primer set and kit for various duck-derived pathogenic bacteria. Background technique [0002] R. anatipestifer ( Riemerella Anatipestifer , RA) is a pathogenic bacteria that infects many kinds of poultry such as ducklings, young turkeys, and goslings. It is the most harmful to ducks aged 1 to 8 weeks. It is clinically acute or chronic sepsis. Perihepatitis, air sacculitis, and meningitis are the main types, and some cases have characteristics such as caseous salpingitis, conjunctivitis, and arthritis, commonly known as duck infectious serositis. [0003] Duck colibacillosis is pathogenic Escherichia coli ( Escherichia coli , E.coli) infection, due to the age of infected ducks, resistance, pathogenicity of Escherichia coli, and different infection routes, many different pathological changes and clinical symptoms can occur. In ducklings, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12Q1/10C12Q1/04C12N15/11
CPCC12Q1/686C12Q2537/143Y02A50/30
Inventor 曾振灵仇珍珍蒋红霞瞿颖李亚菲曹长福
Owner SOUTH CHINA AGRI UNIV
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