Multiplex real-time fluorescent quantitative PCR (Polymerase Chain Reaction) rapid diagnosis kit for five porcine diarrhea viruses and application

A real-time fluorescence quantitative and rapid diagnosis technology, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, and resistance to vector-borne diseases, can solve the problems of cumbersome operation, time-consuming and labor-intensive, low sensitivity, etc.

Active Publication Date: 2017-09-22
ZHEJIANG SCI-TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Routine etiological and serological tests are difficult to distinguish and identify these diseases at the same time. At the same time, there are defects such as cumbersome operation

Method used

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  • Multiplex real-time fluorescent quantitative PCR (Polymerase Chain Reaction) rapid diagnosis kit for five porcine diarrhea viruses and application
  • Multiplex real-time fluorescent quantitative PCR (Polymerase Chain Reaction) rapid diagnosis kit for five porcine diarrhea viruses and application
  • Multiplex real-time fluorescent quantitative PCR (Polymerase Chain Reaction) rapid diagnosis kit for five porcine diarrhea viruses and application

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Experimental program
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Effect test

Embodiment 1

[0074] Embodiment 1, plasmid standard product preparation

[0075] Step 1: Primer Synthesis

[0076] The 5 kinds of viral primer sequences designed in the present invention (see Table 1) were synthesized in Shanghai Sangon Biology Technology Service Company, China, and the synthetic amount was 3 OD per primer.

[0077] Step 2: Total viral DNA / RNA extraction

[0078] Put 100uL of each positive sample containing TGEV, GAR, GCR, PEDV and PCV2 into a 1.5ml centrifuge tube, and use the Viral RNA / DNA Extraction Kit MiniBEST Viral RNA / DNA Extraction KitVer.4.0 (TAKARA) according to the product instruction manual Extract to obtain viral RNA / DNA.

[0079] Step 3: cDNA synthesis by reverse transcription

[0080] Reverse transcription reaction: Add 2 μl of the total RNA / DNA template prepared in the previous step to an RNase-free 0.2ml PCR tube, and perform RT according to the operating instructions of TaKaRa One Step RNA PCR Kit (AMV) (Code No.RR024A). -PCR reaction, after the revers...

Embodiment 2

[0085] Embodiment 2, the present invention detects five kinds of virus-specific experiments

[0086] Step 1: Primer Synthesis

[0087] The 5 kinds of viral primer sequences designed in the present invention (see Table 1) were synthesized in Shanghai Sangon Biology Technology Service Company, China, and the synthetic amount was 3 OD per primer.

[0088] Step 2: Specific detection of a single virus in a multiplex system

[0089] Prepare 5 identical reaction solutions according to the EvaGreen multiplex real-time fluorescent quantitative PCR reaction system (i.e., 15 μL of Master Mix, 20×EvaGreen 1 μl, 10 μM TGEV, GAR, GCR, PEDV and PCV2 upstream and downstream primers are 0.5 μL, 0.5 μL, 0.15μL, 0.15μL, 0.4μL), respectively add 1μl of 1.0×10 6 Copies / μl of plasmid standards for TGEV, GAR, GCR, PEDV, and PCV2, plus ddH 2 O to a total volume of the reaction system of 20 μl; the reaction was carried out on an ABI 7300 fluorescence quantification instrument, and the reaction para...

Embodiment 3

[0091] Embodiment 3, the present invention detects five kinds of virus stability experiments

[0092] Step 1: Primer Synthesis

[0093] With the first step in embodiment 1.

[0094] Perform the following operations on TGEV, GAR, GCR, PEDV and PCV2 respectively:

[0095] Take 6 copies of each virus 1.0×10 6 Copies / μl of plasmid standard and 2 negative controls (DEPC-H 2 0), repeated experiments were carried out within and between batches, respectively. The intra-batch repeat test is to repeat the 3 samples in the same real-time fluorescence three times; the inter-batch repeat test is to carry out the real-time fluorescent quantitative PCR test in 3 different time experiments (interval 3 days). The real-time fluorescent quantitative PCR reaction system and reaction procedure are shown in the second step of the above-mentioned embodiment 2.

[0096] The CV values ​​of the inter-batch and intra-batch repeated tests of the five viruses are all less than 4%, and the invention h...

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Abstract

The invention discloses a multiplex real-time fluorescent quantitative PCR (Polymerase Chain Reaction) rapid diagnosis kit for five porcine diarrhea viruses. The rapid diagnosis kit comprises five pairs of virus specific primers, fluorescent quantitative PCR reaction liquid, a positive reference substance, a negative reference substance and a quantitative PCR standard substance. The invention further discloses application of the kit to multiplex real-time fluorescent quantitative detection of five porcine diarrhea virus infection; and the five porcine diarrhea viruses refer to TGEV, GAR, GCR, PEDV and PCV2.

Description

technical field [0001] The invention belongs to the field of viral nucleic acid detection, in particular to a multiplex real-time fluorescent quantitative PCR rapid diagnostic kit for five porcine diarrhea viruses, which can simultaneously detect porcine epidemic diarrhea virus (Porcine epidemicdiarrhea virus, PEDV), porcine infectivity Gastroenteritis virus (Transmissible gastroenteritis virus, TGEV), porcine rotavirus type A (Porcine group A rotaviruses, GAR), porcine rotavirus type C (Porcine group C rotaviruses, GCR), porcine circovirus type 2 (Porcine circovirus 2, PCV2) five kinds of porcine diarrhea virus, suitable for rapid quantitative detection of five kinds of porcine diarrhea virus in clinical and scientific research. Background technique [0002] In recent years, porcine diarrhea has occurred frequently and on a large scale worldwide, resulting in heavy economic losses (Chae et al, 2000, Sun et al, 2012). Epidemic diarrhea has occurred in various parts of my co...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/701C12Q2561/113C12Q2563/107C12Q2545/114C12Q2537/143Y02A50/30
Inventor 姜永厚刘高鹏
Owner ZHEJIANG SCI-TECH UNIV
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