Triple RT-PCR detection kit

A technology of RT-PCR and detection kits, applied in the field of triple RT-PCR detection kits, can solve the problems of cumbersome operation, aerosol pollution, complicated process, etc., and achieve convenient and fast operation, high sensitivity and strong specificity Effect

Inactive Publication Date: 2015-07-22
SOUTHWEST UNIVERSITY FOR NATIONALITIES +1
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Problems solved by technology

However, virus isolation is time-consuming and labor-intensive and difficult; fluorescent antibody detection and immunohistochemical methods are highly non-specific and require fluorescence microscopy for observation; ELISA pathogen detection method is widely used, but the process is more complicated than PCR method; conventional RT -PCR can only detect one pathogen at a time and needs to perform PCR again after reverse transcription of RNA, which is cumbersome and easy to cause aerosol pollution

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Embodiment Construction

[0033] A triple RT-PCR detection kit, the kit is composed of A box for virus RNA extraction and B box for RT-PCR detection, A box is equipped with buffer RG 9 mL, buffer RF 3 mL , 8 mL buffer RD, 12 mL buffer RW, 3 mL RNA eluent, 8 mL 50-fold TAE electrophoresis buffer, 10 μL staining solution, 30 μL loading buffer, RNase-free adsorption column, 10 collection tubes, 10 centrifuge tubes and 10 0.2 mL PCR tubes, box B contains 220 μL of amplification reaction solution and 10 μL of enzyme system; the amplification reaction solution includes The PCR primer set of virus and porcine A group rotavirus, described primer set is made up of three sets of primer sequences, and the primer sequence that is used for porcine epidemic diarrhea virus detection is respectively SEQ ID NO: 1 and SEQ ID NO: 2; The primer sequences for porcine transmissible gastroenteritis virus detection are SEQ ID NO: 3 and SEQ ID NO: 4; the primer sequences for porcine group A rotavirus detection are SEQ ID NO: 5...

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Abstract

The invention belongs to the field of biological detection and in particular relates to a triple RT-PCR detection kit. Three pairs of specific primers are respectively designed according to PEDV M gene, TGEV N gene and GAR VP 7 gene, wherein the sizes of the amplified fragments are 199bp, 499bp and 333bp respectively; a user can directly judge by naked eyes according to electrophoretic results. The kit is high in specificity, high in repeatability and convenient and quick to operate; single infection or mixed infection can be definitely diagnosed in 4-6 hours generally; the double purposes of diagnosing and identifying can be achieved. The kit is higher in sensitivity; the minimum detection concentration of porcine epidemic diarrhea virus is 4.3 ng per microliter; the minimum detection concentration of porcine transmissible gastroenteritis virus is 3.2 ng per microliter; the minimum detection concentration of rotavirus diarrhea of porcine group A is 6.1 ng per microliter.

Description

technical field [0001] The invention belongs to the field of biological detection, in particular to a triple RT-PCR detection kit. Background technique [0002] The main symptoms of porcine transmissible gastroenteritis virus (PEDV) infection are vomiting, watery diarrhea with severe malodor, weight loss and severe dehydration. The mortality rate of pigs over 5 weeks old is low, and the disease is mild in adult pigs, which may cause loss of appetite and diarrhea, and lactating sows may appear agalactia. However, piglets under the age of 2 weeks are most susceptible to infection, and the morbidity and mortality are high, and death may be caused by starvation, dehydration and acidosis. This kit is suitable for auxiliary diagnosis of porcine epidemic diarrhea virus. [0003] The main symptoms of porcine epidemic diarrhea virus (TGEV) infection are watery diarrhea, or vomiting after eating; sick pigs have normal or slightly high body temperature, depression, loss of appetite o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/70C12Q1/686C12Q2600/16
Inventor 岳华汤承王善普李秀梅陈新刚王方圆姜良张晓辉王俊峰
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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