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Multi-PCR detection kit and detection method for duck-origin common bacteria

A technology for detection kits and detection methods, applied in biochemical equipment and methods, measurement/inspection of microorganisms, resistance to vector-borne diseases, etc., can solve problems such as batch detection, cumbersome fabrication, long detection time, etc., and achieve shortening The effect of detection time, specificity, simple operation and great economic benefits

Active Publication Date: 2011-09-14
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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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 takes a long time to detect, is cumbersome to perform, and cannot be tested in batches

Method used

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  • Multi-PCR detection kit and detection method for duck-origin common bacteria
  • Multi-PCR detection kit and detection method for duck-origin common bacteria
  • Multi-PCR detection kit and detection method for duck-origin common bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. Establishment of a multiplex PCR detection kit for common bacteria from ducks

[0034] Prepare the duck plague bacteria multiplex PCR detection kit according to the following composition:

[0035] Contains 10× reaction buffer, DNA polymerase, dNTP (2.5mM each), 50% Glycerol, and 3 pairs of primers whose sequences are:

[0036] RA DnaB P1: AAACTCAGGCAAAGGTGGCAC (SEQ ID NO. 1)

[0037] RA DnaB P2: TGTATGGTAGTTTTGATGCTTTCAA (SEQ ID NO. 2)

[0038] E. coli phoA P1: CGATTCTGGAAATGGCAAAAG (SEQ ID NO. 3)

[0039] E. coli phoA P2: CGTGATCAGCGGTGACTATGAC (SEQ ID NO. 4)

[0040] Salm invA P1: AACCAGCAAAGGCGAGCAG (SEQ ID NO. 5)

[0041] Salm invA P2: CAATACGATGCTGTTATCGTCCAG (SEQ ID NO. 6)

[0042] Establishment of Multiplex PCR Reaction System and Reaction Conditions

[0043] The present invention optimizes conditions such as the proportioning concentration of each primer, the concentration of glycerol, and the amount of Taq DNA polymerase, and the finally determined react...

Embodiment 2

[0054] Embodiment 2: Multiplex PCR method is to the detection of Riemerella anatipestifer, escherichia coli and Salmonella wild strain in the laboratory (can finish within 3 hours)

[0055] 130 strains of Riemerella anatipestifer, 68 strains of Escherichia coli and 28 wild strains of Salmonella preserved in the laboratory were detected by using the established multiplex PCR method.

[0056] (1) Preparation of bacterial DNA template

[0057] Single colonies of Riemerella anatipestifer, Escherichia coli and Salmonella on TSA / LB were inoculated into TSB or LB liquid medium respectively, and shaken at 37 °C. Then transfer again at 1:100, shake the bacteria until OD600=0.6-0.8, take 0.5mL bacterial solution into 1.5ml Eppondorf tubes, boil at 100°C for 5 minutes, cool at room temperature, and set aside at -20°C. The mixture of three known bacteria, Riemerella anatipestifer, Escherichia coli and Salmonella, was boiled as a positive control; in addition, deionized water was used ins...

Embodiment 3

[0070] Embodiment 3: multiplex PCR is to the detection of clinical sample (can finish within 5 hours)

[0071] Six samples were obtained from brain and liver tissues from ducks infected with Riemerella anatipestifer, E. coli or Salmonella in the laboratory. The liver and brain tissues of 18 dead ducks were collected from the diseased duck farm and suspected to be infected by bacteria (9 ducks in total).

[0072] (1) Preparation of DNA template

[0073] A small amount of liver and brain tissues of dead ducks were taken aseptically, and genomic DNA was extracted with QiAamp? DNA mini kit as a template. Standby at -20°C. The mixture of three known bacteria, Riemerella anatipestifer, Escherichia coli and Salmonella, was boiled as a positive control; in addition, deionized water was used instead of DNA template as a negative control.

[0074] (2) Prepare a multiplex PCR detection reaction system according to the following composition:

[0075] Reaction Component

Volu...

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Abstract

The invention belongs to the technical field of biological detection, and in particular relates to a multi-polymerase chain reaction (PCR) detection kit and a multi-PCR detection method for duck-origin common bacteria. The multi-PCR detection kit for the duck-origin common bacteria comprises three pairs of primers, namely a first pair of primers RADnaBP1 and RADnaBP2, a second pair of primers E.coliphoAP1 and E.coliphoAP2 and a third pair of primers SalminvAP1 and SalminvAP2, wherein the RADnaBP1 has a nucleotide sequence shown as SEQ ID No.1; the RADnaBP2 has the nucleotide sequence shown as SEQ ID No.2; the E.coliphoAP1 has the nucleotide sequence shown as SEQ ID No.3; the E.coliphoAP2 has the nucleotide sequence shown as SEQ ID No.4; the SalminvAP1 has the nucleotide sequence shown as SEQ ID No.5; and the SalminvAP2 has the nucleotide sequence shown as SEQ ID No.6. By the kit and the method, riemerella anatipestifer, escherichia coli and salmonella can be quickly, conveniently, specifically and sensitively detected; and the kit and the method are easy to popularize on a large scale, can be used for bacterial identification, disease diagnosis and epidemiological survey and have wide market prospects and better economic benefits.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a multiple PCR detection kit and a detection method for duck-derived common bacteria. Background technique [0002] Riemerella anatipestifer, Escherichia coli and salmonellosis are three bacterial infectious diseases that endanger the duck industry most seriously. [0003] Riemerella anatipestifer (RA) disease is a contagious disease mainly affecting ducks, turkeys and other birds. So far, there are 21 RA serotypes (type 1-21) that have been confirmed. The main pathological changes of the disease are fibrinous pericarditis, perihepatic inflammation, air sacculitis, meningitis, caseous salpingitis, etc., with high incidence and high mortality rate (mortality rate 1%-80%, even as high as 90%). Endured ducks are mostly stiff ducks, and their growth is retarded, causing great economic losses. [0004] Duck colibacillosis is caused by pathogenic Escherichia...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCY02A50/30
Inventor 于圣青胡青海韩先干丁铲仇旭升宋翠萍
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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