Amphimorphic FQ-PCR detection reagent kit for identifying African swine fever and swine fever virus wild strains

A technology of African swine fever virus and FQ-PCR applied in the field of molecular biology

Pending Publication Date: 2019-08-30
HENAN CENT FOR ANIMAL DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no report on the double FQ-PCR met...

Method used

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  • Amphimorphic FQ-PCR detection reagent kit for identifying African swine fever and swine fever virus wild strains
  • Amphimorphic FQ-PCR detection reagent kit for identifying African swine fever and swine fever virus wild strains
  • Amphimorphic FQ-PCR detection reagent kit for identifying African swine fever and swine fever virus wild strains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 is used to identify the design of primers and probes for ASFV, CSFV dual TaqMan MGB FQ-PCR detection and the establishment of detection methods

[0050] 1.1 Materials

[0051] 1.1.1 Positive standards and strains

[0052] ASFV and CSFV positive standard samples were amplified by PCR and RT-PCR by the Henan Animal Disease Prevention and Control Center respectively, and the target genes were cloned into the pGEM-T Easy vector to obtain ASFV and CSFV positive standard samples; other controls Viruses, bacteria or positive recombinant plasmids were provided by Henan Animal Disease Prevention and Control Center.

[0053] 1.1.2 Instruments and reagents

[0054] Fully automatic nucleic acid extraction instrument, product of Thermo Company of the United States; fluorescent PCR instrument, product of ABI Company of the United States; PCR amplification instrument, product of Biometra Company of Germany; gel imaging analysis system, product of Alpha Innotech Company of ...

Embodiment 2

[0100] Example 2 Differentiate ASFV, CSFV double TaqMan MGB FQ-PCR detection kit

[0101] The double TaqMan MGB FQ-PCR detection kit of the present invention includes ASFV detection primer pair and probe (SEQ ID NO: 1-3), CSFV detection primer pair and probe (SEQ ID NO: 4-6).

[0102] The detection kit also includes one or more of PrimeScript RT Master Mix, dNTPs, reverse transcriptase, Taq DNA polymerase, PCR reaction buffer, positive standard, negative control and the like.

[0103] The specific application of the detection kit is as follows:

[0104] (1) Sample requirements

[0105] 1. Apply proper technique to collect samples.

[0106] 2. The sediment and suspended matter in the sample may affect the test results and should be removed by centrifugation.

[0107] 3. Samples should not be placed at room temperature for more than 6 hours after processing and collection; if not tested within 6 hours, the samples should be placed in a refrigerator at 2-8°C; if they need to b...

Embodiment 3

[0131] Example 3 Distinguish the application of ASFV, CSFV dual TaqMan MGB FQ-PCR detection technology

[0132] The dual TaqMan MGB FQ-PCR detection method for distinguishing ASFV and CSFV established according to the present invention is compared with the conventional PCR detection method for ASFV and CSFV established before, with ASFV and CSFV positive standards as positive controls; sterilized PBS solution as negative In contrast, 10 clinical suspected ASFV or CSFV infection samples (sample numbers are: 1~10) adopt the method provided by the present invention and common PCR / RT-PCR (reverse transcription PCR) detection to carry out laboratory detection respectively, and these The detection results of the two methods were compared and analyzed, and compared with the sequencing results. The results are shown in Table 3. Result shows, adopt the double TaqMan MGB FQ-PCR method detection of distinguishing ASFV provided by the present invention, CSFV, 3 parts are ASFV positive, 5 ...

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Abstract

The invention provides an amphimorphic FQ-PCR detection reagent kit for identifying African swine fever and swine fever virus wild strains. A P72 gene of ASFV and a 5'UTR noncoding region of CSFV arerespectively used as an amplification target area, a pair of specific primers and a TaqMan MGB probe (SEQ ID NO:1-6) are designed, a real-time fluorescent quantitation PCR(FQ-PCR) technique is used, and identification and detection of ASFV and CSFV are realized. The detection reagent kit provided by the invention is suitable for detecting viral nucleic acid in samples of serum, spleen, lymph nodes, tonsil, kidney and the like of suspected ASFV or CSFV infected pigs, the sensitivity can reach 1.0*10<1>copy/[mu]L, and the detection reagent kit does not have any cross reactions with other pathogens which are liable to be in mixed infection with the ASFV and the CSFV or of which the infection symptoms are similar such as PRRSV, PRV, PCV2, PPV, JEV and HPS.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a double FQ-PCR detection kit for identifying African swine fever and wild strains of swine fever virus. Background technique [0002] African swine fever (ASF) is a highly contagious disease caused by African swine fever virus (ASFV). Disorder, accompanied by severe thrombocytopenia and lymphopenia, infected animals usually die within 10d to 14d after infection, and the case fatality rate can reach 100%. The ASFV genome is double-stranded DNA with a size of 170-190kb. Viral genes can encode more than 200 kinds of proteins, including 54 kinds of structural proteins. P72 is a structural protein and is the main component of the viral capsid. The sequence of the P72 gene is conserved, and the same conserved sequence is found among different strains. [0003] Classical swine fever (CSF) is a contagious disease of pigs caused by swine fever virus (CSFV), with high mortality. The cl...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2537/143C12Q2561/101C12Q2563/107
Inventor 闫若潜班付国王淑娟王东方刘影赵美雪杨海波刘毅宋丹赵雪丽谢彩华马震原王翠王华俊刘梅芬柴茂王英华
Owner HENAN CENT FOR ANIMAL DISEASE CONTROL & PREVENTION
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