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Real-time fluorescence quantitative PCR detection primers and probe for SRV (simian type D retrovirus), detection kit and detection method

A technology of real-time fluorescence quantification and detection kit, which is applied in the field of bioengineering to achieve the effects of low cost, high sensitivity and specificity

Active Publication Date: 2017-05-31
广东蓝岛生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have their limitations in terms of specificity, sensitivity, and early diagnosis

Method used

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  • Real-time fluorescence quantitative PCR detection primers and probe for SRV (simian type D retrovirus), detection kit and detection method
  • Real-time fluorescence quantitative PCR detection primers and probe for SRV (simian type D retrovirus), detection kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Design of detection primers and detection probes

[0024] Use Vector NTI Suite software to analyze SRV-1 virus, SRV-2 virus, SRV-3 virus and SRV / D-T virus gene conservative sequence, and then use Primer Express software to design monkey SRV virus detection primers and detection probes. The designed detection primers and detection probes are as follows:

[0025] Upstream primer (SRV-F): 5'-CTGGTCAGCCAATGACGGGTA-3' (shown in SEQ ID NO. 1), downstream primer (SRV-R): 5'-CGCCTGTCTTAGGTTGGAGTGA-3' (shown in SEQ ID NO. 2 ); The amplified target gene fragment is shown in SEQ ID NO.3.

[0026] SRV virus probe: 5'-AGAGAGTGACATTTCTCACTAAC-3' (as shown in SEQ ID NO. 4), the 5'end has a fluorescent reporter group FAM, and the 3'end has a fluorescent quenching group TAMRA.

Embodiment 2

[0027] Example 2: Extraction of SRV proviral DNA

[0028] 1 mL of blood was taken from the monkey femoral vein with a 5 mL sterile syringe, and EDTA was used for anticoagulation. Use the universal genomic DNA extraction kit and follow the instructions to extract genomic DNA from peripheral blood.

Embodiment 3

[0029] Example 3: Detection primer and detection probe specificity test

[0030] Real-time fluorescent quantitative PCR reaction system 20μL, including: template DNA 5μL, 10×TaqMan buffer 2μL, 5mMMgCl 2 1.5μL, 2.5U / μL HotStart DNA polymerase 1μL, 2.5mmol / L dNTPs 1μL, 20μmol / LSRV virus probe 0.25μL, 20μmol / L SRV-F and SRV-R each 0.25μL, sterilized ultrapure 8.75μL of water. The reaction program is 95°C for 30s; 95°C for 5s, 61°C for 31s, 50 cycles.

[0031] Specificity test:

[0032] Take foam virus, SIV, SRV-1, SRV-2, SRV-3 and SRV / D-T positive samples and normal cynomolgus peripheral blood to extract genomic DNA. Perform real-time fluorescent quantitative PCR amplification according to the above-mentioned real-time fluorescent quantitative PCR reaction system. The results showed that the amplification results of foamy virus and SIV positive samples and normal cynomolgus monkey peripheral blood were negative, and the amplification results of SRV-1, SRV-2, SRV-3 and SRV / D-T positi...

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Abstract

The invention discloses real-time fluorescence quantitative PCR detection primers and probe for SRV (simian type D retrovirus), a detection kit and a detection method. The detection primers and the probe are designed on the basis of SRV-1, SRV-2, SRV-3 and SRV / D-T gene conserved sequences, and the real-time fluorescence quantitative PCR detection method for the SRV is established. Compared with other detection methods, the detection method has higher sensitivity and specificity, can detect a large quantity of samples simultaneously, is low in cost and is suitable for large-scale SRV detection for a monkey field. Reliable background information can be provided for scientific research of selection of macaca fascicularis and macaques on the basis of high SRV detection rate, and monkeys carrying the SRV can be discovered and isolated in advance.

Description

[0001] Technical field: [0002] The invention belongs to the technical field of bioengineering, and specifically relates to a real-time fluorescent quantitative PCR detection primer, probe, detection kit and detection method of monkey SRV virus. [0003] Background technique: [0004] Cynomolgus monkeys and rhesus monkeys are widely used in pathology, experimental zoology and pharmacology research. In the process of research, experimental monkeys carrying D retrovirus (simian type D retrovirus, SRV) will seriously interfere with experimental results. At the same time, SRV virus has Highly infectious and highly pathogenic. In the past, the detection of SRV virus mainly used virus isolation and serological methods, such as enzyme-linked immunoassay (Elisa) and immunofluorescence. These methods have their limitations in specificity, sensitivity, and early diagnosis. In the previous serological diagnosis, it was reported in the relevant literature that the positive serological test of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/702C12Q2561/113C12Q2563/107
Inventor 陈臻毅梁自豪张剑凯
Owner 广东蓝岛生物技术有限公司
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