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RT-LAMP detection primer pairs, kit and detection method for infectious haematopoietic necrosis virus (IHNV)

A technology of RT-LAMP and hematopoietic organ necrosis, applied in the field of molecular biology, can solve the problems of long detection period, not adapting to the requirements of the times, etc., and achieve the effects of simple operation, high sensitivity and simple identification.

Inactive Publication Date: 2016-02-03
东莞出入境检验检疫局检验检疫综合技术中心 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] It generally takes more than 15 days to obtain the results of culturing and isolating infectious hematopoietic organ necrosis virus, and the detection cycle is too long to meet the requirements of the era of rapid detection at ports and large customs clearance

Method used

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  • RT-LAMP detection primer pairs, kit and detection method for infectious haematopoietic necrosis virus (IHNV)
  • RT-LAMP detection primer pairs, kit and detection method for infectious haematopoietic necrosis virus (IHNV)
  • RT-LAMP detection primer pairs, kit and detection method for infectious haematopoietic necrosis virus (IHNV)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Establishment of Infectious Hematopoietic Necrosis Virus RT-LAMP Detection Kit

[0041] RT-LAMP detection kit for infectious hematopoietic necrosis virus, including RT-LAMP primer set, RT-LAMP reaction solution, Bst DNA polymerase, AMVReverse transcriptase, positive and negative controls.

[0042] (1) RT-LAMP primer design: using the envelope glycoprotein gene of infectious hematopoietic necrosis virus (IHNV) ( glyG Gene, GenBank accession number is DQ164100.1) as the target gene, using the online design software PrimerExplorerversion4 (http: / / primerexplorer.jp / e) to design LAMP primers. The primer sequences are listed in Table 1.

[0043] Table 1 Primer sequence list

[0044]

[0045] (2) RT-LAMP reaction solution: containing 10mMdNTP, 10×ThermoPol reaction buffer, and 150mMMgSO4 aqueous solution, the volume ratio of the three is 8:5:2.

[0046] (3) The positive control is a T vector clone containing the envelope glycoprotein gene fragment of infe...

Embodiment 2

[0048] Example 2 Using a turbidimeter to establish an infectious hematopoietic necrosis virus RT-LAMP detection method:

[0049] Utilize the method for detecting infectious hematopoietic necrosis virus with the kit of embodiment 1, comprise the steps:

[0050] (1) Extraction of RNA from samples to be tested: Extract RNA from samples using a virus RNA extraction kit;

[0051] (2) Constant temperature gene amplification reaction: 25μl reaction system contains: IHNV-F30.2μM, IHNV-B30.2μM, IHNV-FIP1.6μM, IHNV-BIP1.6μM, IHNV-LF0.8μM, IHNV-LB0.8μM, RT-LAMP reaction solution 12.5μl, AMV reverse transcriptase 1U, Bst 8U of DNA polymerase, 1~100ng of RNA to be tested, make up to 25μl with DEPC water; set up positive control and negative control; mix the prepared PCR tube and centrifuge, and react at 63~65℃ for 60~90min, and in 80°C for 2 minutes;

[0052] (3) Result judgment: judge the amplification result by observing the turbidity change of the precipitate in the reaction tube...

Embodiment 3

[0053] Example 3 ESE-Quanttube scanner detection

[0054] The kit is the same as in Example 1 except that the chromogenic agent (SYBRGreenI) not in Example 1 is added.

[0055] Use the above kit to detect infectious hematopoietic necrosis virus (IHNV) by the following method:

[0056] (1) Extraction of RNA from the sample to be tested: Extract and purify the RNA from the sample to be tested using a viral RNA extraction kit;

[0057] (2) Constant temperature gene amplification reaction: 25μl reaction system contains: IHNV-F30.2μM, IHNV-B30.2μM, IHNV-FIP1.6μM, IHNV-BIP1.6μM, IHNV-LF0.8μM, IHNV-LB0.8μM, RT-LAMP reaction solution 12.5μl, AMV reverse transcriptase 1U, Bst DNA polymerase 8U, 10×SYBRGreenI0.5μl, RNA to be tested 1~100ng, make up to 25μl with DEPC water; set positive control and negative control; mix the prepared PCR tube and centrifuge, react at 65℃ for 60min, and Continue at 80°C for 2 minutes;

[0058] (3) Result judgment: Place the reaction tube in ESE-Q...

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Abstract

The invention discloses RT-LAMP detection primer pairs, a detection kit and a detection method for infectious haematopoietic necrosis virus (IHNV). The detection primer pairs comprises a pair of outer primers, a pair of inner primers and a pair of loop primers; the detection kit comprises primer liquid, reaction liquid, DNA polymerase, reverse transcriptase and control; and the detection kit also can contain a color developing agent. The detection method comprises the following steps: extracting to-be-detected virus RNA, and amplifying a sample RNA template at the temperature of 63-65 DEG C by utilizing reverse transcription activity of reverse transcriptase and adopting six specific primers and one DNA polymerase with strain displacement activity, wherein the pg grade of pure virus RNA can be detected; and identifying by adding SYBR Green I ESE-Quant-tube Scanner instrument detection, or utilizing a turbidity meter for observing change of turbidity of sediments in a reaction tube, so as to judge whether to carry out amplification or not and determine whether the to-be-detected sample contains IHNV RNA or not. The RT-LAMP detection primer pairs, the detection kit and the detection method for the IHNV have the advantages of quickness, high efficiency, easy operation, high specificity, high sensitivity, easy identification and applicability to field detection and are applicable to popularization and application.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and relates to a detection method for marine aquaculture, in particular to an RT-LAMP detection primer set, detection kit and detection method for infectious hematopoietic organ necrosis virus (IHNV). Background technique [0002] Infectious hematopoietic necrosis (IHN) is one of the three rhabdovirus diseases that need to be notified according to OIE regulations. IHN was originally endemic in western North America, mainly in anadromous salmon stocks. Infectious hematopoietic necrosis virus (IHNV) infection became an important pathogen of rainbow trout (Oncorhynchus mykiss) cultured in the United States in the 1970s. Then IHN spread to many countries in Western Europe and East Asia along with the fish eggs contaminated with the virus. Rainbow trout is an imported species in these countries, and IHN has caused serious economic losses to rainbow trout farming. Similar to other members o...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2521/107C12Q2531/119
Inventor 陈进会陈珍容黄伟颜其贵邱杨刘建丽赵丽
Owner 东莞出入境检验检疫局检验检疫综合技术中心
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