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A pathogen multiplex pcr detection kit for chicken common bacterial diseases

A detection kit and technology for bacterial diseases, applied in the determination/inspection of microorganisms, methods based on microorganisms, and resistance to vector-borne diseases, etc., can solve the problems of time-consuming, cumbersome detection methods, troublesome pathogenic bacteria and mixed infections, etc. , to achieve good specificity and sensitivity, and great application value

Active Publication Date: 2019-10-25
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the detection of bacterial diseases in farms mainly relies on bacteriological culture, which generally takes 4-7 days. The detection method is cumbersome and time-consuming. It is troublesome to detect and isolate multiple pathogenic bacteria and mixed infections in farms at the same time.
Western blotting, enzyme-linked immunosorbent assay, nucleic acid hybridization technology, PCR technology, etc. are widely used because of their strong specificity, high sensitivity, simple and fast operation, but these methods usually only detect one pathogen at a time

Method used

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  • A pathogen multiplex pcr detection kit for chicken common bacterial diseases
  • A pathogen multiplex pcr detection kit for chicken common bacterial diseases
  • A pathogen multiplex pcr detection kit for chicken common bacterial diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Pathogenic multiplex PCR detection kit for chicken common bacterial diseases and its application method

[0043] A pathogen multiplex PCR detection kit for chicken common bacterial diseases, the kit includes: multiplex PCR reaction solution, positive control, negative control, DNAMarker2000 (100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp; TaKaRaLtd), four pairs of bacteria The specific primer (10uM): rfbE-F (SEQ ID NO.1), rfbE-R (SEQ ID NO.2), invA-F (SEQ ID NO.3), invA-R (SEQ ID NO.4 ), KMT1-F (SEQ ID NO.5), KMT1-R (SEQ ID NO.6), nuc-F (SEQ ID NO.7), nuc-R (SEQ ID NO.8);

[0044] The multiple PCR reaction solution is: Taq DNA polymerase (2-5U / μL), Mg-free 2+ 10× PCR reaction buffer, 25mM Mg 2+ and 10mM dNTPs;

[0045] Described positive control is: Escherichia coli O157:H7 (CICC21530) standard substance, Salmonella (CVCC3375) standard substance, Pasteurella multocida (CVCC44801) standard substance, Staphylococcus aureus (SY1G7) standard substance (purchased from China ...

Embodiment 2

[0073] Design and Screening of Specific Primers of Multiplex PCR Detection Kit for Common Bacterial Diseases in Chickens

[0074] 1. Materials and methods

[0075] 1. Bacterial strains

[0076] Standard strains: Pasteurella multocida CVCC44801, Salmonella enteritidis CVCC 3375, Escherichia coli CICC21530 and Staphylococcus aureus SY1G7 standard strains were purchased from China Microbiology species collection center. 16 strains of bacteria isolated and identified clinically in the laboratory and their serial numbers: avian bacteria Salmonella HZBL08121, Salmonella CMCC50051, Salmonella HZBL08122, Salmonella HZBL08123, Pasteurella multocida HBNKM, Pasteurella multocida HBNK1G1, Staphylococcus aureus GX12G54, cholera Vibrio HS1201, Morganella HZBL08123, Rothella GX1202, Enterococcus faecalis HS12E6, Riemerella anatipestifer ATCC11845, Escherichia coli HZBL08121, Escherichia coli HZBL08122, Staphylococcus pseudointermediate GX12G6, Staphylococcus squirrel GX12G5.

[0077] 2. T...

Embodiment 3

[0100] Specificity and sensitivity of multiplex PCR detection method for four common bacterial disease pathogens in chickens:

[0101] 1. Materials and methods

[0102] 1. The standard bacterial strain is the same as in Example 2.

[0103] 2. According to the method for using the multiplex PCR detection kit described in Example 1, extract the DNA of Pasteurella multocida, Salmonella, Escherichia coli O157:H7 and Staphylococcus aureus standard samples respectively and perform multiplex PCR reactions.

[0104] 3. Specific detection of quadruple PCR reaction

[0105] Select 16 strains of avian-derived bacteria (same as in Example 2) clinically isolated in the laboratory to extract DNA, and perform multiplex PCR detection.

[0106] 4. Sensitivity detection of quadruple PCR reaction

[0107] Measure the DNA concentration of the four strains (Pasteurella multocida, Salmonella, Escherichia coli O157:H7 and Staphylococcus aureus) and dilute them so that the initial concentration is...

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Abstract

The invention discloses a pathogen multiplex PCR (polymerase chain reaction) detection kit for chicken common bacterial diseases. The kit comprises a PCR reaction solution, specific primers for Pasteurella multocida, Salmonella, Escherichia coli O157:H7 and Staphylococcus aureus, positive controls (standard substances of Pasteurella multocida, Salmonella, Escherichia coli O157:H7 and Staphylococcus aureus, or positive plasmids of Pasteurella multocida KMT1 gene, Salmonella invA gene, Escherichia coli O157:H7 rfbE gene and Staphylococcus aureus nuc gene), a negative control (sterile enzyme-free double distilled water), and a DNA marker. The quadruplex PCR is utilized to quickly, accurately and synchronously detect the four pathogenic bacteria; the kit has the advantages of high sensitivity and high specificity; the lower detection limit reaches 0.01ng / mu L; and the accuracy is up to 92.6%.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a pathogen multiplex PCR detection kit for chicken common bacterial diseases, and also relates to a using method of the kit. Background technique [0002] Escherichia coli O157:H7, Salmonella, Staphylococcus aureus and Pasteurella multocida are common pathogens of bacterial diseases during chicken growth. They not only seriously endanger the development of my country's poultry industry, but also Escherichia coli O157:H7, Salmonella, and Staphylococcus aureus can infect humans through food contamination, posing a threat to human health. [0003] Escherichia coli (E.Coli) O157:H7 is a major strain of enterohemorrhagic Escherichia coli, which can cause zoonotic diseases. and human safety are seriously jeopardized. Salmonella (Salmonella) is a zoonotic pathogenic bacteria, which is very easy to produce drug resistance and seriously endangers the aquaculture...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12Q1/10C12Q1/14C12R1/42C12R1/19C12R1/445C12R1/01
CPCC12Q1/686C12Q1/689C12Q2537/143C12Q2545/113C12Q2545/101Y02A50/30
Inventor 谷长勤张晓茜程国富张万坡胡薛英谢长清
Owner HUAZHONG AGRI UNIV
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