Multi-fluorescent quantitative PCR reagent kit capable of synchronously detecting three bovine respiratory pathogens, and multi-fluorescent quantitative PCR detection method capable of synchronously detecting three bovine respiratory pathogens

A multiplex fluorescence quantitative detection kit technology, applied in the field of livestock disease detection, can solve the problems of the multiplex fluorescence quantitative PCR method that has not been reported yet, time-consuming, low sensitivity and accuracy, etc., and achieves high sensitivity, strong specificity, good repetitive effect

Inactive Publication Date: 2019-07-16
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the detection methods for Mycobacterium bovis, Mycoplasma bovis, and Klebsiella bovis mostly use bacteriological examination methods such as smear microscopy, bacterial isolation and identification, and conventional immunological methods, which have low sensitivity and accuracy. Time-consuming and other issues
The PCR method can also be used for the detection of the above three pathogens, but the multiplex fluorescent quantitative PCR method for the effective detection of these three pathogens at the same time has not been reported yet

Method used

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  • Multi-fluorescent quantitative PCR reagent kit capable of synchronously detecting three bovine respiratory pathogens, and multi-fluorescent quantitative PCR detection method capable of synchronously detecting three bovine respiratory pathogens
  • Multi-fluorescent quantitative PCR reagent kit capable of synchronously detecting three bovine respiratory pathogens, and multi-fluorescent quantitative PCR detection method capable of synchronously detecting three bovine respiratory pathogens
  • Multi-fluorescent quantitative PCR reagent kit capable of synchronously detecting three bovine respiratory pathogens, and multi-fluorescent quantitative PCR detection method capable of synchronously detecting three bovine respiratory pathogens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1 Primer and probe design

[0073] According to the specific sequence 229bp sequence of Mycobacterium bovis registered on GenBank (accession number: AJ003103), the uvrC gene of Mycoplasma bovis (accession number: AF003959) and the khe gene of Klebsiella bovis (accession number: AF293352) using Oligo7 .0 software designed specific primers and TaqMan probes, and selected the following specific primers and TaqMan probes.

[0074] Using the genomic DNA of Mycobacterium bovis, Mycoplasma bovis and Klebsiella bovis as templates and the corresponding specific primers to amplify, the corresponding specific product size fragments were displayed after electrophoresis, such as figure 1 .

[0075] The amplified products were purified, cloned and then sequenced. The sequencing results were blasted on NCBI. As a result, each pair of primers was consistent with the theoretical sequence in the database, and there was only a single base difference. This difference may be due...

Embodiment 2

[0079] Example 2 Simultaneous detection of multiple fluorescent quantitative PCR detection kits for three bovine respiratory pathogens

[0080] In addition to the primers and TaqMan probes described in Example 1, the above kit also includes the following reagents: PremixEx Taq TM (2×), template DNA and with sterile ddH2O.

[0081] Further, the amplification reaction system is 20 μL, including the following: Premix Ex TaqTM (2×) 10 μL, upstream and downstream primers (10 μM / L) each 0.5 μL, probe (10 μM / L) each 0.5 μL, template DNA 2 μL, Make up to 20 µL with sterile ddHO water.

[0082] Amplification was carried out according to the following reaction parameters: 95°C for 600s, 95°C for 10s, 60°C for 10s, 72°C for 20s, 45 cycles, 95°C for 10s, 65°C for 60s, 97°C for 1s, 37°C for 30s.

Embodiment 3

[0083] Example 3 Simultaneous detection of multiple fluorescent quantitative PCR detection methods for three bovine respiratory pathogens

[0084] Using the primers and TaqMan probes in Example 1, and the kit in Example 2, multiplex fluorescent quantitative PCR detection was performed.

[0085](1) Establish a standard curve: respectively construct recombinant plasmids carrying Mycobacterium bovis, Mycoplasma bovis and Klebsiella bovis specific sequences (specific sequences are as shown in Example 1), prepare DNA standard items, and multiply by 10 times respectively Serially diluted to 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 Copy / μL, with different concentrations of standard products as templates, fluorescent quantitative PCR was carried out under the guidance of the primers in Example 1 and TaqMan probes. The amplification reaction system of fluorescent quantitative PCR is 20 μL, including the following: Premix Ex TaqTM (2×) 10 μL, upstream and downstream primers (10 μ...

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Abstract

The invention belongs to the technical field of livestock disease detection, and discloses a multi-fluorescent quantitative PCR reagent kit capable of synchronously detecting three bovine respiratorypathogens as well as a multi-fluorescent quantitative PCR detection method capable of synchronously detecting the three bovine respiratory pathogens, wherein the three bovine respiratory pathogens arerespectively mycobacterium bovis, mycoplasma bovis and Klebsiella pneumoniae. According to the multi-fluorescent quantitative PCR detection method capable of synchronously detecting the three bovinerespiratory pathogens, 3 groups of specific primers and probes are designed and synthesized on basis of a specific sequence 229bp of the mycobacterium bovis, a uvrC gene of the mycoplasma bovis and aKhe gene of the Klebsiella pneumoniae, thereby establishing the multi-fluorescent quantitative PCR detection method capable of synchronously detecting the mycobacterium bovis, the mycoplasma bovis andthe Klebsiella pneumoniae which are associated with respiratory diseases in cattle. The multi-fluorescent quantitative PCR detection method is capable of synchronously detecting the 3 bacteria; and sensitivity, repeatability and specificity tests have proven that the detection method is high in sensitivity, strong in specificity and relatively good in repeatability.

Description

technical field [0001] The invention relates to the technical field of livestock disease detection, in particular to a multiplex fluorescent quantitative PCR kit and method for simultaneously detecting three bovine respiratory pathogens, wherein the three bovine respiratory pathogens are Mycobacterium bovis, Mycoplasma bovis, Kreb pneumoniae primary bacteria. Background technique [0002] Bovine respiratory disease is an important disease in cattle production, which is characterized by high morbidity and mortality, so it will bring serious economic losses to cattle production, and also cause serious damage to the cattle industry all over the world . Bovine respiratory diseases may be related to one or more pathogens, among which Mycobacterium bovis, Mycoplasma bovis, and Klebsiella bovis are three very common and important pathogens. Bovine tuberculosis is mainly caused by Mycobacterium bovis infection, which is mainly transmitted through the respiratory tract. Cows suffe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/10C12Q1/06C12N15/11C12R1/32C12R1/35C12R1/22
CPCC12Q1/6851C12Q1/689C12Q2600/16C12Q2531/113C12Q2537/143C12Q2561/101C12Q2545/114
Inventor 周向梅吴文学董玉慧王杰
Owner CHINA AGRI UNIV
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