Primers, probes and detection kits for detection of porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus

A porcine epidemic diarrhea and detection kit technology, which is applied in biochemical equipment and methods, microbial measurement/inspection, material stimulation analysis, etc., can solve problems such as inability to diagnose, insufficient sensitivity, and many false positives, and save time and cost, and the effect of improving detection efficiency

Inactive Publication Date: 2011-12-14
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Abstract
  • Description
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Problems solved by technology

Although the above method can differentiate and diagnose the two diseases, due to the limitation of the experimental method, the detection time is too long, and the diagnosis cannot be made in the early stage of virus infection.
In recent years, with the development of molecular biology, the application of reverse transcription-polymerase chain reaction (RT-PCR) in the detection of PEDV and TGEV has become more and more extensive. Compared with traditional methods, RT-PCR method is more Sensitive, fast, and convenient, but complicated steps, many false positives, lack of accurate quantification, and insufficient sensitivity hinder the application of RT-PCR in actual clinical practice

Method used

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  • Primers, probes and detection kits for detection of porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1, the composition of kit

[0055] 1. The composition of the kit

[0056] 1) RNA extraction reagent; Qiagen RNA extraction kit (QIAamp Viral RNA Mini kit, cat.no 52906);

[0057] 2) RNA standard product: it is the RNA fragment transcribed in vitro of porcine transmissible gastroenteritis virus, and its corresponding DNA sequence is SEQ ID No: 9 in the sequence table; and the RNA fragment transcribed in vitro of porcine epidemic diarrhea virus, and its corresponding DNA sequence is The sequence is the sequence listing SEQ ID No: 12;

[0058] 3) Real-time quantitative RT-PCR reaction solution, which includes: 1× RT-PCR buffer containing dNTP, 0.2 μM sense primer, 0.2 μM antisense primer and 0.4 μM fluorescent probe for detecting porcine transmissible gastroenteritis virus, Detection of porcine epidemic diarrhea virus sense primer 0.2μM, antisense primer 0.2μM and fluorescent probe 0.4μM, Taq DNA polymerase 2U, reverse transcriptase 5U, 1× fluorescent quantita...

Embodiment 2

[0076] Embodiment 2 utilizes RNA standard substance to establish standard curve

[0077] According to the concentration of RNA (TGEV-N-T7 / PEDV-N-T7) that has been measured above, use the formula: (6.02 x 1023 copies / mole) x (concentration g / ml) x10 -3 / (MW g / mol) = copy / μL, calculate the copy number of TGEV-N-T7 and PEDV-N-T7 respectively, common ten-fold dilution, with 10 3 ~10 7 The copy / μl RNA is used as a template, and the reaction is carried out on the ABI7300 fluorescent quantitative PCR instrument. The reaction system is 20 μL, of which 10 μL of 2x One Step RT-PCR Buffer, TaKaRa Ex Taq TM HS (5U / μL) 0.4μL, PrimeScript TM RT Enzyme MixⅡ 0.4μL, PEDV-F and TGEV-F (10μM) each 0.4μL, PEDV-R and TGEV-R (10μM) each 0.4μL, PEDV-Probe and TGEV-Probe (10μM) each 0.8μL, ROX Reference Dye (50x) 0.4 μL, template RNA 2 μL, DDW to 25 μL. The reaction conditions were 50°C for 30 minutes, followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds. The data was analyzed ...

Embodiment 3

[0078] Embodiment 3, the specificity test of kit

[0079] 1 Materials, see Table 2:

[0080] Virus source porcine epidemic diarrhea virus Harbin Veterinary Research Institute Veken Company porcine transmissible gastroenteritis virus Harbin Veterinary Research Institute Veken Company porcine respiratory and reproductive disorder syndrome virus Shanghai Haili Biological Pharmaceutical Co., Ltd. porcine circovirus Separation, identification and preservation of pig disease group of Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences porcine pseudorabies virus Wuhan Keqian Animal Biological Products Co., Ltd. porcine parvovirus Bought from China Veterinary Drug Control Institute swine fever virus Nanjing Tianbang swine fever cell vaccine porcine encephalitis virus Hunan Yahua Seed Industry Co., Ltd. Biopharmaceutical Factory porcine rotavirus Separation, identificat...

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Abstract

The invention discloses a primer, a probe and a detection kit for detecting porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus. The detection primers and probes are SEQ ID NO: 1 to SEQ ID NO: 6 in the sequence table, wherein the sequences SEQ ID NO: 1 and SEQ ID NO: 2 are sense primers and antisense primers for detecting porcine transmissible gastroenteritis virus respectively, and the sequence SEQ ID NO: 3 For detecting the fluorescent probe of porcine transmissible gastroenteritis virus, sequence SEQIDNO: 4 and SEQIDNO: 5 are sense primer and antisense primer for detecting porcine epidemic diarrhea virus respectively, and sequence SEQIDNO: 6 is the detection primer of porcine epidemic diarrhea virus fluorescent probe. The invention also provides detection kits for porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus. The primers and probes selected by the present invention have very strong specificity. The total viral RNA extracted from porcine diarrhea does not need to be transcribed into cDNA first. The synthesis of the first strand of cDNA and double PCR are completed in one step, and two viruses can be detected at one time. ,Improve efficiency.

Description

technical field [0001] The invention relates to primers, probes and detection kits for detecting porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus. Background technique [0002] Porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV) and porcine transmissible gastroenteritis virus (Transmissible gastroenteritis virus, TGEV) are the main enteroviruses that cause diarrhea in piglets. Viruses, single-stranded positive-sense RNA viruses, can cause atrophy or shortening of intestinal villi. They all have similar transmission routes and clinical symptoms. Infected pigs all show watery diarrhea, vomiting, dehydration and high mortality of newborn piglets. Occurs widely around the world. Since the clinical manifestations, pathological changes, and epidemiology of infectious gastroenteritis and epidemic diarrhea are very similar, but the two pathogens have no common antigenicity, cannot cross immunity, and have mixed infections, so the t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor 袁秀芳王一成徐丽华李军星刘邓
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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