Primer and probe for identifying dual real-time fluorescence quantitative PCR (polymerase chain reaction) of A type and B type cattle pasteurella multocida and detection method

A technology for real-time fluorescence quantification and Pasteurella, which is applied in the direction of microbial-based methods, biochemical equipment and methods, and microbial determination/inspection, can solve the problem of not identifying Type A and Type B Pasteurella multocida, Weak cross-protection of different serotypes to achieve the effect of ensuring safety and rationality, high sensitivity and good repeatability

Inactive Publication Date: 2018-08-17
JINYUBAOLING BIO PHARMA CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Chinese Patent Publication No. CN107164557A discloses primers and applications of a dual real-time fluorescent quantitative PCR method for simultaneously detecting Pasteurella multocida and its capsule type A, but it does not carry out Type A and Type B multocida Identification of bacilli
Although this type of vaccine plays an important role in the control of bovine Pasteurellosis, the cross-protection against different serotypes is weak

Method used

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  • Primer and probe for identifying dual real-time fluorescence quantitative PCR (polymerase chain reaction) of A type and B type cattle pasteurella multocida and detection method
  • Primer and probe for identifying dual real-time fluorescence quantitative PCR (polymerase chain reaction) of A type and B type cattle pasteurella multocida and detection method
  • Primer and probe for identifying dual real-time fluorescence quantitative PCR (polymerase chain reaction) of A type and B type cattle pasteurella multocida and detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1. is designed to be used for the primer of the dual real-time fluorescent quantitative PCR of identification A type and B type Bovine Pasteurella multocida Objects and TaqMan Probes

[0063] Since the nucleotide sequences of Type A and Type B bovine Pasteurella multocida are relatively large, it is key to find better different site specificity for the detection of Type A and Type B bovine Pasteurella multocida. Retrieve from NCBI's nucleic acid database GenBank (http: / / www.ncbi.nlm.nih.gov) to obtain the regional genes of type A bovine Pasteurella multocida hyaD-hyaC (Pasteurella multocida capsule biosynthesis gene cluster, GenBank number: AF067175.2) and the sequence of the region gene of Pasteurella multocida capsulebiosynthesis gene cluster, complete sequence GenBank: AF169324) of Type B bovine Pasteurella multocida bcbD were compared with DNAMan software, and designed according to primers and TaqMan probes In principle, select the regional gene of Pa...

Embodiment 2

[0077] Embodiment 2. Use primers of the present invention and TaqMan probe to carry out double-acting to type A and type B bovine Pasteurella multocida Heavy real-time fluorescent quantitative PCR differential detection

[0078] Step 1. Extract genomic DNA from the sample

[0079] Genomic DNA is extracted from the bacterial cultures of Type A and Type B bovine Pasteurella multocida (for obtaining standard items and obtaining positive controls) and samples to be tested, and the specific method includes the following steps ( Blood & Tissue Kit, QUAIGEN Company):

[0080] (1) QUAIGEN kit preparation: According to the kit instructions, prepare (96%-100%) ethanol in advance and add absolute ethanol of specified concentration to reagent Buffer AW1 and Buffer AW2.

[0081] (2) Add 100 μL of sample to a 1.5 mL centrifuge tube, add 20 μL proteinase K and 180 μl BufferATL, and vortex to mix.

[0082] (3) In the 1.5mL centrifuge tube in step (2), add 200μL Buffer AL to the mixtur...

Embodiment 3

[0112] Embodiment 3. Specificity experiment

[0113] Utilize QUAIGEN kit according to the method for embodiment 2 to A type, B type, D type, E type and F type Pasteurella multocida, Brucella, bovine type A clostridium, bovine type B clostridium, Bovine Clostridium type C, bovine Clostridium type D, porcine circovirus type 2 (PCV2), rhinotracheitis virus (IBRV) extract DNA, against foot-and-mouth disease type A, foot-and-mouth disease type O, foot-and-mouth disease Asia type, bovine viral diarrhea virus (BVDV), bovine epidemic fever virus extract RNA, simultaneously take the DNA that type A and type B bovine Pasteurella multocida bacterial culture extracts as a positive control, and use non-enzyme water as a negative control, primers of the present invention and TaqMan probe Under the guidance of the needle, double real-time fluorescent quantitative PCR detection was performed, and the PCR reaction system and reaction conditions were referred to in Example 2 to verify the spe...

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Abstract

The invention relates to a primer and a probe for identifying dual real-time fluorescence quantitative PCR of A type and B type cattle pasteurella multocida and a detection method, and belongs to thetechnical field of biological detection. The primer and the probe are designed and obtained by taking the sequences of hyaD-hyaC area genes of A type cattle pasteurella multocida and bcbD area genes of B type cattle pasteurella multocida as the detection sequences. By utilizing the primer and the probe, detection operation is simple and convenient, the specificity is high, the sensitivity is high,the repeatability is good, and the primer and the probe can play the role in identifying, screening and controlling production quality of vaccine strains of A type and B type cattle pasteurella multocida.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a primer and a probe for dual real-time fluorescent quantitative PCR for distinguishing Type A and Type B bovine Pasteurella multocida, a detection kit comprising the primer and the probe, and The method uses the primers and probes or the detection kit to detect type A and type B bovine Pasteurella multocida for non-disease diagnosis purposes. Background technique [0002] Pasteurellosis [0003] Pasteurellosis is a kind of infectious disease caused by Pasteurella multocida (PM), which can be found in various livestock and humans. Mainly cause hemorrhagic septicemia or infectious pneumonia in animals. Animals can infect each other, and humans can be infected by animal bites. Pasteurellosis in cattle has a short incubation period, rapid transmission and high mortality, which often causes significant economic losses to animal husbandry. [0004] Pasteur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6851C12Q1/689C12Q2600/16
Inventor 陈君彦张宸王艳杰王秀明王云凌刘建奇武槿贤刘国英魏学峰韩四娥黄海碧史文瑞徐丽媛范秀丽
Owner JINYUBAOLING BIO PHARMA CO LTD
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