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Fluorescent quantitative PCR primers and probes for porcine epidemic diarrhea viruses

A swine epidemic diarrhea, fluorescence quantitative technology, applied in biochemical equipment and methods, microbial determination/inspection, microorganisms, etc., can solve the problems of pig industry losses, lack of fluorescence quantitative differential diagnosis methods, etc., to achieve primer specificity and high sensitivity, simple operation effect

Active Publication Date: 2014-01-15
WENS FOODSTUFF GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005]At present, PEDV still occurs on a large scale in mainland China, which has brought huge losses to China's pig industry
Some scholars have conducted a detailed analysis of the popular PEDV strains and their genetic variation (Pan Y et al., Isolation and characterization of a variant porcine epidemic diarrhea virus in China[J]. Virol J. 2012, Sep 12 ;9:195.), found that the S gene between the mutant strain and the classic PEDV strain is quite different, which can be used as a characteristic gene to distinguish the two strains, but there is no fluorescent quantitative differential diagnosis method for this Appear

Method used

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  • Fluorescent quantitative PCR primers and probes for porcine epidemic diarrhea viruses
  • Fluorescent quantitative PCR primers and probes for porcine epidemic diarrhea viruses
  • Fluorescent quantitative PCR primers and probes for porcine epidemic diarrhea viruses

Examples

Experimental program
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Effect test

Embodiment 1

[0034] (1) RNA extraction of the sample to be tested: 200 μL cell culture medium (infected with the self-isolated strain CHGD-01, which has been identified as a mutated PEDV, and other mutated strains can also be used), 200 μL of cell culture medium (infected with the self-isolated strain strain ShQT, which has been identified as classic PEDV, and other classic strains can also be used), 100 mg of intestinal disease material and 100 mg of feces (the disease material and feces are unknown whether the virus is infected), and DMEM (cell culture medium, without PEDV, as a negative control) as the sample to be tested, the following operations were performed respectively:

[0035] Add 1mL of PBS buffer for thorough grinding, repeat freeze-thaw at -20°C for 3 times, centrifuge at 8000 rpm for 10 min, take 200 μL of supernatant, add 0.2mL of chloroform, shake and mix for 15 seconds, and then store at room temperature (15°C-30°C) After standing for 2-3 minutes, centrifuge at 12000g (2°...

Embodiment 2

[0044] Example 2 specificity test

[0045] Porcine transmissible gastroenteritis virus, porcine rotavirus, porcine reproductive and respiratory disorder syndrome The culture fluid of porcine circovirus, porcine circovirus, classical swine fever virus, and porcine pseudorabies virus is specifically detected, and the method, primers and probes of Example 1 are used. As a result, there is no amplification curve except the positive control, and the positive disease material After PCR amplification, there are amplification curves in HEX (strain ShQT) and FAM (strain CHGD-01) groups respectively (see figure 2 ).

Embodiment 3

[0046] Embodiment 3 Sensitivity test

[0047] Standard positive plasmids (pMD-S1 and pMD-S2) containing the S gene, 191bp amplified by the PCR involved in this patent (use the cDNA extracted from the mutant strain ShQT as a template, and use SEQ ID NO: 1~2 as primers for PCR , the amplified product sequence is shown in SEQ ID NO.5) and 179bp (with the cDNA extracted from the classical strain CHGD-01 as a template, and SEQ ID NO: 1~2 are primers for PCR, the amplified product sequence is shown in SEQ ID NO .6) was connected to the cloning vector pMD18-T (Dalian Bao Biological Engineering Co., Ltd.), and its concentration was determined to be 3.94×10 10 copies / μL and 2.99×10 10 copies / μL.

[0048] The positive plasmids pMD-S1 and pMD-S2 were diluted 10 times with the prepared sterile double distilled water as the template, and 2 μL was taken for each dilution as the template, and the detection was carried out according to the method in Example 1, and the amplification curv...

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Abstract

The invention discloses fluorescent quantitative PCR (polymerase chain reaction) primers and probes for porcine epidemic diarrhea viruses. An upstream primer and a downstream primer are shown as SEQIDNO: 1-2; a probe P1 is shown as SEQIDNO: 3; a probe P2 is shown as SEQIDNO: 4; the 3' end of sequences of the probe 1 and the probe 2 are combined with a fluorescent quenching group; and the 5' end of sequences of the probe 1 and the probe 2 are combined with different fluorescent reporter groups. The primers are relatively high in specificity and sensitivity, can detect out variant PEDV (porcine epidemic diarrhea viruses), and can detect and differentiate a variety of clinical samples so as to quickly differentiate epidemic PEDV strains; the operation is simple and practical.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a dual fluorescent quantitative PCR primer and probe capable of distinguishing variant and classic porcine epidemic diarrhea viruses. Background technique [0002] Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is a porcine intestinal infectious disease caused by porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV) belonging to the Coronaviridae family and the genus Coronaviridae of the order Mandoviridae. Characterized by diarrhea, vomiting and dehydration (Takahashi K, Okada K, Ohshima K.An outbreak of swine diarrhea of ​​a new type associated with coronavirus-like particles in Japan[J].Vet Sci, 1983,45:829-832; Murphy FA, Fauquet C M. smith Report of the ICTV[J], Archives virology suppl,1995,10: 407-409.; Yin Zhen, Liu Jinghua. Animal Virology[M].2 edition. Beijing: Science Press, 1997 . 681-688.). Pigs of various ages can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/70C12Q2531/113C12Q2563/107
Inventor 王东东宋延华周庆丰潘永飞李春梅田小艳卢围廖承球
Owner WENS FOODSTUFF GRP CO LTD
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