Multiple real-time fluorescent quantative PCR rapid diagnosis kit for six porcine viruses

A real-time fluorescence quantitative and rapid diagnosis technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of difficult early detection, cumbersome operation, time-consuming and laborious, etc.

Active Publication Date: 2015-04-29
浙江洪晟生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Multiple real-time fluorescent quantative PCR rapid diagnosis kit for six porcine viruses
  • Multiple real-time fluorescent quantative PCR rapid diagnosis kit for six porcine viruses
  • Multiple real-time fluorescent quantative PCR rapid diagnosis kit for six porcine viruses

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Embodiment 1 kit structure

[0077] see figure 1 , a multiplex real-time fluorescent quantitative PCR rapid diagnostic kit for six porcine viruses, consisting of fluorescent quantitative PCR reaction solution 1, PCV2 virus standard product 2, PPV virus standard product 3, PRRSV virus standard product 4, CSFV virus standard product 5, PEDV virus Standard substance 6, PRV virus standard substance 7, positive control substance 8, negative control substance 9 and box body 10.

[0078] The box body 10 is provided with corresponding container holes for respectively placing quantitative PCR reaction solution tubes, PCV2 virus standard product tubes, PPV virus standard product tubes, PRRSV virus standard product tubes, CSFV virus standard product tubes, and PEDV virus standard product tubes. , PRV virus standard quality control, positive control quality control and negative control quality control. Wherein the fluorescent quantitative PCR reaction solution comprises 3 tubes o...

Embodiment 2

[0086] Embodiment 2 preparation of plasmid standard

[0087] Step 1: Primer Synthesis

[0088] The 6 viral primer sequences (see Table 1) designed by the present invention were synthesized in China Shanghai Sangon Biology Technology Service Company, and the synthesis amount was 2OD per primer.

[0089] Step 2: Total viral DNA / RNA extraction

[0090] Put 10ul of dry powder samples containing PCV2, PPV, PRRSV, CSFV, PEDV and PRV virus sources into 1.5ml centrifuge tubes, and use the MiniBEST Viral RNA / DNA Extraction Kit Ver. .4.0 (TAKARA) extraction to obtain viral RNA / DNA.

[0091] Step 3: Synthesize cDNA by reverse transcription

[0092] Reverse transcription reaction: Add 0.5 μl of the total RNA template prepared in the previous step to an RNase-free 0.2ml PCR tube, and perform RT- PCR reaction, after the reverse transcription reaction is completed, the resulting reaction solution is the cDNA / DNA template.

[0093] Step 4: Common PCR amplification

[0094] PCR reaction ...

Embodiment 3

[0097] Embodiment 3 The present invention detects six kinds of virus-specific experiments

[0098] Step 1: Primer Synthesis

[0099] The 6 viral primer sequences (see Table 1) designed by the present invention were synthesized in China Shanghai Sangon Biology Technology Service Company, and the synthesis amount was 2OD per primer.

[0100] Step 2: Specific detection of a single virus in a multiplex system

[0101] According to the EvaGreen multiplex real-time fluorescent quantitative PCR reaction system, 6 identical reaction solutions were prepared, and 1 μl of 1.0×10 6 Copies / μl of PCV2, PPV, PRRSV, CSFV, PEDV and PRV plasmid standards, that is, the reaction system is 5μl of 10×PCR buffer, 25mM MgCl 2 5μl, 2.5mM dNTP 4μl, 20×EvaGreen 2.5μl, Ex Taq DNA polymerase 2.5U, in which the final concentrations of upstream and downstream primers for PCV2, PPV, PRRSV, CSFV, PEDV and PRV are 0.26, 0.12, 0.14, 0.13, 0.05 and 0.13 respectively μM, 1μl template, add ddH 2 O to the total...

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Abstract

The invention discloses a multiple real-time fluorescent quantative PCR rapid diagnosis kit for six porcine viruses. The kit comprises six pairs of PRC primers; PCV2-F, PCV2-R, PPV-F, PPV-R, PRRSV-F, PRRSV-R, PEDV-F, PEDV-R, PRV-F, PRV-R, CSFV-F and CSFV-R. The kit further comprises a fluorescent quantative PCR reaction liquid, a positive reference substance, a negative reference substance and a quantative PCR standard substance. The kit disclosed by the invention can be used for detecting PCV2, PPV, PRRSV, CSFV, PEDV and PRV from a sample through a PCR reaction at the same time. Compared with conventional common PCR and single fluorescent quantative PCR methods, the kit is simpler and more time-saving and convenient.

Description

technical field [0001] The invention belongs to the field of viral nucleic acid detection, and in particular relates to a rapid multiplex real-time fluorescent quantitative PCR diagnostic kit for six kinds of porcine viruses, which can simultaneously detect porcine circovirus type II (PCV2) and porcine parvovirus (PPV) in the same reaction tube, Porcine reproductive and respiratory syndrome virus (PRRSV), swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV) and pseudorabies virus (PRV) six porcine viruses, suitable for rapid quantification of six porcine viruses in clinical and scientific research detection. Background technique [0002] In recent years, with the development of large-scale and intensive pig farming, the occurrence of multi-pathogen mixed infection and secondary infection in pig herds has become more and more common. Porcine parvovirus (PPV), porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome (PRRSV), pseudorabi...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/6851C12Q1/701C12Q2600/16C12Q2531/113C12Q2537/143C12Q2561/113C12Q2563/107
Inventor 姜永厚饶品彬吴海港
Owner 浙江洪晟生物科技股份有限公司
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