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Multiply-fluorescence PCR (polymerase chain reaction) detection kit for identifying strong and weak strains of mycoplasma gallisepticum

A technology of Mycoplasma gallisepticum and multiple fluorescence, which is applied in the directions of fluorescence/phosphorescence, determination/inspection of microorganisms, recombinant DNA technology, etc., can solve the problems of long time consumption, low sensitivity, inability to determine the infection content of Mycoplasma gallisepticum, etc. Sensitivity and specificity, effects of high amplification

Active Publication Date: 2012-07-25
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these differential detection methods are time-consuming, low-sensitivity, and conventional PCR requires the use of a PCR machine and gel electrophoresis, which cannot determine the content of Mycoplasma gallisepticum infection

Method used

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  • Multiply-fluorescence PCR (polymerase chain reaction) detection kit for identifying strong and weak strains of mycoplasma gallisepticum
  • Multiply-fluorescence PCR (polymerase chain reaction) detection kit for identifying strong and weak strains of mycoplasma gallisepticum
  • Multiply-fluorescence PCR (polymerase chain reaction) detection kit for identifying strong and weak strains of mycoplasma gallisepticum

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1, the design of primer and probe and the preparation of kit

[0046] 1. Design of primers and probes

[0047]According to the sequence of Mycoplasma gallisepticum in the gene bank, a comparison is made to determine the gene conservation region for strong and weak viruses (genbank: 335524-335633 nucleotides of CP001872) and the gene conservation region for weak viruses (genbank: nucleotides 335633 of CP001873 603585-603714 nucleotides), using Primer Express3.0 software to design primer pairs and probes, the primer sequences are:

[0048] MG Strong and Weak Virus Universal Forward Primer (MG1): 5'-TTGCTAACCGCAAGGAAGC-3' (Sequence 1)

[0049] MG strong and weak general reverse primer (MG2): 5'-CTCCATAGAAAGGAGGTAATCCA-3' (sequence 2)

[0050] MG Strong and Weak Virus Universal TaqMan Probe (MG3):

[0051] 5'-CCGGTGATTGGAGTTAAGTCGTAACAAG-3' (SEQ ID NO: 3)

[0052] The 5' end of the MG strong and weak TaqMan probe MG3 is labeled with the reporter fluorescent d...

Embodiment 2

[0065] Example 2, the application of primers and its kit in the detection of strong and weak strains of Mycoplasma gallisepticum by multiplex fluorescent PCR

[0066] 4 virulent strains of MG (S6, A5969, K-2221(383T), K-1501-S) and 2 attenuated strains of MG (F2F10, K810) are recorded in the following reference 1;

[0067] MG attenuated vaccine strain (MG-F-vaccine) and chicken infectious laryngotracheitis virus (ILTV) were provided by China Veterinary Drug Administration; MG attenuated vaccine (MG-F-vaccine-Guangdeng) was provided by Guangdong Yongshun Bioengineering Co., Ltd. ;

[0068] Records of Mycoplasma gallisynovii (MS-K1415), Mycoplasma turkey (MM-TACC), Mycoplasma Iowa (MI-I695), Newcastle disease virus (NDV-Laseta) and chicken infectious bronchitis virus (IBV Mass 41) In reference 2 below;

[0069] Salmonella avian (C 7913 ) is described in Reference 3 below; avian influenza virus (AIV H9) is described in Reference 4 below.

[0070] References: [1] Han Wang, A.A...

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Abstract

The invention discloses a multiply-fluorescence PCR (polymerase chain reaction) detection kit for identifying strong and weak strains of mycoplasma gallisepticum. Primers used consists of a first primer, a second primer, a third primer and a fourth primer, the sequences of the first primer, the second primer, the third primer and the fourth primer in the sequence table are respectively a sequence1, a sequence 2, a sequence 4 and a sequence 5. The experiment indicates that on one hand, high amplification and excellent specificity of the PCR technology and quick sensitivity of the fluorescencedetection technology are sufficiently used in the agent and detection method; on the other hand, primer pairs universal to the strong and weak strains of the mycoplasma gallispticum and primer pairs special for weak strains and probes are assembled into a fluorescent PCR system for multiply-fluorescence PCR detection, and mutual interference is avoided, and detection sensitivity and specificity are improved further.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a multiplex fluorescent PCR detection kit for identifying strong and weak strains of mycoplasma gallisepticum. Background technique [0002] Mycoplasma gallisepticum (MG) is the main pathogen of chronic respiratory diseases in chickens and other poultry. Infected chickens often have symptoms such as rales, cough, and runny nose, which can cause a decrease in chicken growth rate, a decrease in broiler carcass quality and Egg production rate, fertilization rate and hatching rate decline, etc., causing greater economic losses to the poultry industry. MG exists widely in all countries in the world, and the infection rate of MG in chicken flocks in my country is very high. Guo Rui et al. separated mycoplasma from 162 chickens suffering from respiratory diseases from 38 chicken farms in Hubei and Henan, and the result was 59 isolates. Was identified as Mycoplasma gallisepticum. Deng Xianw...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11G01N21/64
Inventor 谢芝勋罗思思谢丽基邓显文刘加波谢志勤庞耀珊范晴
Owner GUANGXI VETERINARY RES INST
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