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 direction of fluorescence/phosphorescence, measurement/testing of microorganisms, recombinant DNA technology, etc., can solve the problems of low sensitivity, time-consuming, uncertain content of Mycoplasma gallisepticum infection, etc., and achieve high Amplification, enhanced sensitivity and specificity effects
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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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