RT-LAMP (reverse transcription and loop-mediated isothermal amplification) detection primer group, RT-LAMP detection kit and RT-LAMP detection method for simian immunodeficiency virus

An immunodeficiency virus, RT-LAMP technology, applied in the field of molecular biology, can solve the problems of complex operation procedures and high false positive rate of WB method, and achieve the effect of mild conditions, high specificity and low probability

Active Publication Date: 2017-05-10
GUANGDONG LAB ANIMALS MONITORING INST
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The false positive rate of ELISA method and IFA method is high and the operation procedure of WB method is complicated, which limits its application scope to a certain extent.

Method used

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  • RT-LAMP (reverse transcription and loop-mediated isothermal amplification) detection primer group, RT-LAMP detection kit and RT-LAMP detection method for simian immunodeficiency virus
  • RT-LAMP (reverse transcription and loop-mediated isothermal amplification) detection primer group, RT-LAMP detection kit and RT-LAMP detection method for simian immunodeficiency virus
  • RT-LAMP (reverse transcription and loop-mediated isothermal amplification) detection primer group, RT-LAMP detection kit and RT-LAMP detection method for simian immunodeficiency virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Establishment of RT-LAMP detection kit for monkey immunodeficiency virus

[0041] RT-LAMP (Reverse Transcription and Loop-mediated Isothermal Amplification) test kit for monkey immunodeficiency virus, including RT-LAMP primer set, RT-LAMP reaction solution, BstDNA polymerase, reverse Recording enzyme, positive control and negative control.

[0042] (1) RT-LAMP primer design: Take the main core protein gene sequence (gag) of Simian Immunodeficiency Virus (SIV) as the target gene, and use software to design SIV-specific RT-LAMP primers. The primer sequence is shown in Table 1.

[0043] Table 1. SIV specific RT-LAMP primer sequence list

[0044]

[0045] (2) RT-LAMP reaction solution: 10mM dNTP, 10×ThermoPol reaction buffer, 150mM MgSO 4 Aqueous solution.

[0046] (3) Bst DNA polymerase and AMV reverse transcriptase.

[0047] (4) The positive control is a plasmid DNA containing a fragment of the core protein gene (gag) of the monkey immunodeficiency virus (SIV). The prepa...

Embodiment 2

[0050] Example 2 RT-LAMP detection method of monkey immunodeficiency virus

[0051] Use the above kit to detect Simian Immunodeficiency Virus (SIV) according to the following methods:

[0052] (1) Extraction of RNA from the sample to be tested: Extract the RNA of the sample to be tested with a viral RNA extraction kit from Megi Bio;

[0053] (2) Reverse transcription loop mediated isothermal amplification reaction:

[0054] 25μl reaction system contains: SIV-F3 0.2μM, SIV-B3 0.2μM, SIV-FIP 1.6μM, SIV-BIP 1.6μm, SIV-LF 0.8μM, SIV-LB 0.8μM, RT-LAMP reaction solution 12.5μl, BstDNA 8U polymerase, 2.5U AMV reverse transcriptase, 1-100ng RNA to be tested, make up to 25μl with DEPC water; set up positive control and negative control; mix the prepared PCR tube, centrifuge, and react at 65°C for 60min.

[0055] (3) Result judgment: Observe whether the solution becomes turbid by naked eyes or analyze whether there is a ladder-like band by agarose gel electrophoresis; if the solution in the reac...

Embodiment 3

[0056] Example 3 Specific Experiment

[0057] The method of Example 2 was used to detect the Simian Immunodeficiency Virus (SIV) RNA, Simian D-type Retrovirus (SRV) RNA, Simian Lymphocyte-tropic Virus (STLV) RNA, and Simian B Virus (BV) DNA.

[0058] The identification results showed that with the monkey immunodeficiency virus (SIV) RT-LAMP primer set as primers, the solution in the SIVRNA and plasmid DNA reaction tube became turbid, and obvious ladder-like bands appeared after electrophoresis. Negative controls and SRV, STLV, BV, etc. The reaction tube solution did not appear turbid, and the electrophoresis did not appear ladder-like bands, showing that the detection primer and method of the present invention have good specificity. (see figure 1 ).

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Abstract

The invention discloses an RT-LAMP (reverse transcription and loop-mediated isothermal amplification) detection primer group, an RT-LAMP detection kit and an RT-LAMP detection method for a simian immunodeficiency virus. The detection primer group comprises a pair of outer primers, a pair of loop primers and a pair of inner primers. The detection kit comprises the primer group, RT-LAMP reaction liquid, Bst DNA polymerase, reverse transcriptase as well as negative control and positive control. The detection method comprises the following steps: extracting to-be-detected virus RNA; conducting reverse transcription on the RNA under the action of revertase; then, amplifying a sample template by virtue of six specific primers and one Bst DNA polymerase having strand displacement activity at 63-65 DEG C; and judging whether a to-be-detected sample contains the simian immunodeficiency virus (SIV) RNA by observing whether a reaction tube solution becomes muddy or not with naked eyes or performing electrophoresis to analyze whether a ladder exists or not. The detection primer group, the detection kit and the detection method provided by the invention have the advantages of being highly specific, high in sensitivity, rapid and efficient, simple and convenient to operate, easy in result reading and the like, and are suitable for popularization and application in grassroots animal epidemic disease monitoring units and experimental monkey breeding enterprises in remote areas.

Description

Technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a method for detecting related pathogens in experimental animals, and in particular to a RT-LAMP (Reverse Transcription and Loop-mediated Isothermal Amplification) of Simian Immunodeficiency Virus (SIV). Increasing reaction) detection primer set, detection kit and detection method thereof. Background technique [0002] The first SIV, also known as monkey HIV, was discovered in 1985, and several virus strains were subsequently isolated from African monkeys and Asian rhesus monkeys. SIV genetics is mainly divided into five major branches: SIVcpz, SIVsm, SIVmnd, SIVsyk and SIVagm. SIV has host-dependent evolution characteristics, so that it does not cause disease in the natural host. For example, SIVsm infects black and white face monkeys, and SIVagm infects Africa. Green monkeys, SIVcpz infection of chimpanzees, etc. will not cause the latter to develop disease, but when SIV sp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/703C12Q2531/119C12Q2545/113C12Q2521/107
Inventor 刘助红王静练月晓黄碧洪郭鹏举张钰黄韧
Owner GUANGDONG LAB ANIMALS MONITORING INST
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