General real-time fluorescent RT (Reverse Transcription)-PCR (Polymerase Chain Reaction) method and kit for detecting porcine reproductive and respiratory syndrome virus
A technology for respiratory syndrome and pig reproduction, applied in the direction of fluorescence/phosphorescence, biochemical equipment and methods, and microbial measurement/inspection, can solve the problems of insufficient sensitivity, specificity or timeliness, and achieve good reliability, The effect of strong specificity and high sensitivity
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Embodiment 1
[0026] Construction of standard RNA
[0027] (1) Experimental reagents
[0028] Restriction enzyme Pst I and its buffer, DL2000Marker were purchased from TaKaRa Company;
[0029] Ligation kit, Taq DNA polymerase, AMV reverse transcriptase, RNase inhibitor, dNTPs were purchased from Promega;
[0030] DNA gel recovery kit, purchased from OMEGA company;
[0031] High-efficiency Escherichia coli competent cells DH5α were purchased from Beijing Quanshijin Biotechnology Co., Ltd.;
[0032] Primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.;
[0033] Other biochemical reagents are imported subpackages or domestic analytical pure.
[0034] (2) Experimental equipment
[0035] TGRANDIENT PCR instrument: purchased from HYBAID Company; DYFIII2 type electrophoresis instrument: purchased from Bio-Rad Company;
[0036] UVP gel imaging analysis system: purchased from Gene Company; Steril Biological safety cabinet: purchased from BAKER Company; co...
Embodiment 2
[0050] Extraction of sample RNA
[0051] According to conventional methods, using QIAGEN The Mini Kit kit (purchased from QIAGEN Company) was used to extract the RNA of porcine reproductive and respiratory syndrome virus (JXA1 strain).
Embodiment 3
[0053] Screening test of specific primer and probe combinations for porcine reproductive and respiratory syndrome virus general-purpose real-time fluorescent RT-PCR method
[0054] (1) Experimental reagents
[0055] One Step PrimeScript TM RT-PCR Kit (Perfect Real Time) kit was purchased from TaKaRa Company;
[0056] Porcine reproductive and respiratory syndrome virus universal TaqMan MGB probe and primers were synthesized by Shanghai Jikang Biotechnology Co., Ltd.
[0057] (2) Experimental equipment
[0058] Applied Biosystems 7500 Real-Time PCR System
[0059] (3) Experimental process
[0060] ① Design of primers and probes for porcine reproductive and respiratory syndrome virus (JXA1 strain)
[0061] The specific primers for porcine reproductive and respiratory syndrome virus refer to: oligonucleotide chains with a length of about 18 to 20 bases, specific nucleotides in the highly conserved region of the porcine reproductive and respiratory syndrome Nsp7 gene Fragmen...
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