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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  • Abstract
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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,

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

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Example Embodiment

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

[0045] 1) FMCA primer:

[0046] After screening a large number of designed primers, it was found that the primer base sequences SEQ ID NO: 1 and SEQ ID NO: 2 have the best effect on the FMCA method to distinguish the HP-PRRSV vaccine GDr180 strain from the wild strain. The base sequence 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 probe base sequence SEQ ID NO: 3 has the best effect on the FMCA method to distinguish the HP-PRRSV vaccine GDr180 strain from the wild strain. The base sequence is shown below.

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

Example Embodiment

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

[0053] FMCA analysis of standard samples

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

[0055] Take HP-PRRSV vaccine GDr180 strain separately, add 3mL PBS hydrochloric acid buffer solution to dissolve, take HP-PRRSV wild virus GD strain cell culture solution, respectively take 200μL according to TAKARA company’s MiniBEST Viral RNA / DNA Extraction Kit Ver.4.0 instruction manual for nucleic acid extract.

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

[0057] In order to verify the identification ability of the designed primers and probes on actual samples, this study used HP-PRRSV vaccine strain GDr180 and HP-PRRSV wild virus GD strain as standard samples for FMCA analysis. The nucleic acid extracted from the above-mentioned standard samples was used as a nucleic acid template, and FMCA ampli...

Example Embodiment

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

[0067] FMCA analysis of clinical samples

[0068] 1) Extract viral nucleic acid from the sample: Take 2g samples of pig lung tissue suspected of being infected with HP-PRRSV, add 3mL PBS hydrochloric acid buffer solution for grinding, centrifuge the ground homogenate at 4000×g for 8 min, and aspirate the centrifuged supernatant Store the solution at -80℃ for later use; or 200μL of serum sample; tissue sample homogenate and serum sample for nucleic acid extraction according to the instructions of MiniBEST Viral RNA / DNA Extraction Kit Ver.4.0 of TAKARA Company.

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

[0070] With the standard GDr180 as the positive control, when the absolute value of...

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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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