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Primers, probe and method for rapidly distinguishing HP-PRRS (High pathogenic porcine reproductive and respiratory syndrome) vaccine strain GDr180 from HP-PRRS wild strain

A wild strain, rapid technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of difficulty in popularization and use, loss of pig farming, low cure rate, etc., and achieve repeatability Good, fast detection speed, good specificity

Active Publication Date: 2016-08-31
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] In the summer of 2006, an outbreak of highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) broke out in southern China. Spread to all parts of the country, causing serious losses to my country's pig industry
Although this method has high sensitivity and specificity, it involves two MGB probes, which is expensive and difficult to popularize.

Method used

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  • Primers, probe and method for rapidly distinguishing HP-PRRS (High pathogenic porcine reproductive and respiratory syndrome) vaccine strain GDr180 from HP-PRRS wild strain
  • Primers, probe and method for rapidly distinguishing HP-PRRS (High pathogenic porcine reproductive and respiratory syndrome) vaccine strain GDr180 from HP-PRRS wild strain
  • Primers, probe and method for rapidly distinguishing HP-PRRS (High pathogenic porcine reproductive and respiratory syndrome) vaccine strain GDr180 from HP-PRRS wild strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 A primer and probe for rapidly distinguishing the highly pathogenic porcine reproductive and respiratory syndrome live vaccine GDr180 strain from wild strains

[0045] 1) FMCA primers:

[0046] After screening a large number of primers designed, it was found that the base sequences of the primers SEQ ID NO: 1 and SEQ ID NO: 2 have the best effect on the FMCA method for distinguishing the HP-PRRSV vaccine GDr180 strain from the wild strain. As follows.

[0047] P1: 5'-GGTTGACATGCTGACTTG-3' (SEQ ID NO: 1),

[0048] P2: 5'-CACTGAACCAATGGTGAG-3' (SEQ ID NO: 2).

[0049] 2) Probe

[0050] After screening a large number of designed probes, it was found that the base sequence of the probe, SEQ ID NO: 3, had the best effect on the FMCA method for distinguishing the HP-PRRSV vaccine GDr180 strain from wild strains, and its base sequence is shown below.

[0051] Probe P: 5'-FAM-CCCAAGGATGATTCTCGAGACACCG-BHQ1-3' (SEQ ID NO: 3), or its nucleotide reverse complement. ...

Embodiment 2

[0052] Example 2 A detection method for rapidly distinguishing the highly pathogenic porcine reproductive and respiratory syndrome live vaccine GDr180 strain from wild strains

[0053] FMCA Analysis of Standard Samples

[0054] 1) Extraction of PRRSV standard nucleic acid:

[0055] Take HP-PRRSV vaccine GDr180 strain, add 3mL PBS hydrochloric acid buffer solution to dissolve, take HP-PRRSV wild virus GD strain cell culture solution, take 200μL respectively, and perform nucleic acid extraction according to the instructions of TAKARA's MiniBEST Viral RNA / DNA Extraction KitVer.4.0 extract.

[0056] 2) FMCA operation steps for positive standard samples

[0057] In order to verify the ability of the designed primers and probes to identify actual samples, HP-PRRSV vaccine strain GDr180 and HP-PRRSV wild virus GD strain were used as standard samples for FMCA analysis. The above-mentioned standard samples were used to extract nucleic acids as nucleic acid templates, and the FMCA am...

Embodiment 3

[0066] Example 3 A method for rapidly distinguishing the highly pathogenic porcine reproductive and respiratory syndrome live vaccine GDr180 strain from wild strains

[0067] FMCA analysis of clinical samples

[0068] 1) Extract viral nucleic acid from samples: take 2g of pig lung tissue samples suspected to be infected with HP-PRRSV, add 3mL of PBS hydrochloric acid buffer solution for grinding, centrifuge the ground homogenate at 4000×g for 8 min, and absorb the centrifuged supernatant or 200 μL of serum samples; tissue sample homogenate and serum samples were subjected to nucleic acid extraction according to the instructions of TAKARA’s MiniBEST Viral RNA / DNA Extraction Kit Ver.4.0.

[0069] 2) Using the extracted viral nucleic acid as a template, perform FMCA analysis with the method described in Example 2 above, and the result analysis method is as follows:

[0070] With the standard GDr180 as the positive control, when the absolute value of the melting peak ∆Tm value be...

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Abstract

The invention discloses primers, probe and method for rapidly distinguishing an HP-PRRS (High pathogenic porcine reproductive and respiratory syndrome) vaccine strain GDr180 from an HP-PRRS wild strain. The method combines a real-time PCR (Polymerase Chain Reaction) technology with an MCA (Melting Curve Analysis) technology, and according to a difference of Tm values of a melting curve, the strain GDr180 and the wild strain are identified; the primers, the probe and the method are simple to operate, i.e. only the probe needs to be added before a PCR reaction; a detection speed is high and flux is high, i.e. the entire operating process only needs 3 hours, and time required for parting is greatly shortened; cost is relatively low, i.e. the identifying and detecting aims can be fulfilled only by one common probe; accuracy is high, specificity is good, repeatability is good, analysis can be accurately and rapidly carried out under the high flux, and the primers, the probe and the method are beneficial to popularization and application in clinical practice.

Description

technical field [0001] The invention relates to a method for distinguishing live virus vaccine strains from wild strains, in particular to a detection method for quickly distinguishing highly pathogenic porcine reproductive and respiratory syndrome live vaccine GDr180 strains from wild strains and primers and probes thereof. Background technique [0002] In the summer of 2006, an outbreak of highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) broke out in southern China. Spread to all parts of the country, cause serious loss to my country's pig industry. HP-PRRS is an acute and highly lethal disease caused by a variant strain of porcine reproductive and respiratory syndrome virus (PRRSV), which is called highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). At present, my country mainly adopts comprehensive prevention and control measures based on vaccine immunization, and implements compulsory immunization for pigs. The hig...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/701C12Q2563/107C12Q2527/107C12Q2561/113C12Q2545/114
Inventor 刘志成张建峰沈海燕张春红孙俊颖康桦华
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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