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VHSV (viral haemorrhagic septicaemia virus) visual nucleic acid strip detection primer group and detection method

A technology of viral hemorrhage and nucleic acid test strips, applied in biochemical equipment and methods, methods based on microorganisms, microorganisms, etc., can solve problems such as inapplicability of rapid differential diagnosis, difficulty in satisfying rapid detection, time-consuming and laborious detection, etc., to achieve Improve identification work efficiency, low application cost, and intuitive results

Active Publication Date: 2016-05-04
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The result of cell culture isolation VHSV method is reliable, but the detection cycle is long, which is not suitable for rapid differential diagnosis; the traditional virus cell culture method and serological method are limited by their own characteristics, the detection is time-consuming and laborious, and it is difficult to meet the needs of rapid detection

Method used

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  • VHSV (viral haemorrhagic septicaemia virus) visual nucleic acid strip detection primer group and detection method
  • VHSV (viral haemorrhagic septicaemia virus) visual nucleic acid strip detection primer group and detection method
  • VHSV (viral haemorrhagic septicaemia virus) visual nucleic acid strip detection primer group and detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: VHSV test strip detection

[0037] PCR product test strip detection test

[0038] Take 5 μL of the amplification product and drop it on the sample pad of the test strip, and add 3 drops of buffer solution at the same time, observe the quality control line and detection line after 10 minutes, and judge the negative and positive results.

[0039] The amplified product is dropped on the sample pad of the test strip for detection, and a red band appears in the detection band area, and there is no red band in the blank control ( figure 1 ).

Embodiment 2

[0040] Embodiment 2: the specificity of nucleic acid test strip method

[0041] VHSV281-F and VHSV281-R primers were used for RT-PCR amplification of VHSV, IHNV, and SVCV RNA and nucleic acid test strip detection, as can be seen from the detection results ( figure 2 ), only VHSV amplified products had specific red bands, and IHNV, SVCV and blank control had no bands at the detection line position, showing the good specificity of the detection method.

Embodiment 3

[0042] Embodiment 3: the sensitivity of nucleic acid test strip method

[0043] Extract and determine the concentration of VHSV pseudovirus RNA, respectively diluted to 6.6, 33, 66, 6.6 × 10 2 , 6.6×10 3 , 6.6×10 4 , 6.6×10 5 , 6.6×10 6 , 6.6×10 7 Copy / μL, carry out RT-PCR, detect with nucleic acid test strips, it can be seen from the results ( image 3 ), the detection limit of the nucleic acid test strip method can reach 33 copies / μL.

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Abstract

The invention discloses a VHSV visual nucleic acid strip detection primer group and detection method. The sequences of primers are VHSV281-F:5'-FITC-CAGGCGTTGTCCGTGCTTCT-3', 5' terminal marking FITC and VHSV281-R:5'-biotin-TCCCCGAGTTTCTTGGTGATGTA-3',5' terminal marking biotin. The detection method comprises the following steps of (1) RT-PCR amplification; (2) PCR product test strip detection test. The method has the advantages that the operation is convenient, the specificity and the sensitivity are high, the result is intuitive, stable, reliable and environment-friendly and the like and is suitable for VHSV rapid screening of the port quarantine laboratory, the identification working efficiency of the entry and exit port is improved, the requirement for fast customs clearance on import and export trades can be well met, a used RT-PCR product detection test strip is a general test strip and can be widely used for development of PCR diagnostic reagents, no instruments and equipment such as an electrophoresis apparatus and a transilluminator are used in the detection method, the application cost is low, and the method has a great promoting effect on popularization and application of the PCR detection method to base laboratories.

Description

Technical field: [0001] The invention relates to a primer set and a detection method for visual nucleic acid test strip detection of viral hemorrhagic sepsis virus (VHSV). Background technique: [0002] Viral hemorrhaic septicaemia (viralhaemorrhaic septicaemia, VHS) is a severe infectious disease caused by rhabdoviruses in dozens of species of fish such as salmon, trout, pike and turbot. ~100%. The disease is mainly prevalent in the northern hemisphere, and it is a disease required by the World Organization for Animal Health (OIE) to be declared. my country lists it as a second-class disease. The VHSV genome is a single-stranded negative-strand RNA whose linear genome encodes five proteins, including: nucleocapsid protein (N protein), two structural proteins (M1 and M2 proteins), glycoprotein (G protein) and RNA polymerase (L protein). VHSV particles are about 180nm long and 70nm wide. Its genome is about 12k bases in length. [0003] At present, the commonly used meth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2565/625
Inventor 房保海崔淑华赵丽青厉艳赵鹏刘鑫
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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