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Simian SRV virus real-time fluorescent quantitative PCR detection primers, probes, detection kits and detection methods

A real-time fluorescence quantitative and detection kit technology, applied in the field of bioengineering, to achieve the effect of low cost, high sensitivity and specificity

Active Publication Date: 2020-10-16
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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  • Simian SRV virus real-time fluorescent quantitative PCR detection primers, probes, detection kits and detection methods
  • Simian SRV virus real-time fluorescent quantitative PCR detection primers, probes, detection kits and detection methods

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1: Design of detection primers and detection probes

[0019] Vector NTI Suite software was used to analyze the conserved gene sequences of SRV-1 virus, SRV-2 virus, SRV-3 virus and SRV / D-T virus, and then Primer Express software was used to design detection primers and detection probes for monkey SRV virus. The designed detection primers and detection probes are as follows:

[0020] 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.

[0021] The probe of SRV virus: 5'-AGAGAGTGACATTTCTCACTAAC-3' (as shown in SEQ ID NO.4), with a fluorescent reporter group FAM at the 5' end and a fluorescent quencher group TAMRA at the 3' end.

Embodiment 2

[0022] Embodiment 2: Extraction of SRV proviral DNA

[0023] 1 mL of blood was drawn from the femoral vein of monkeys with a 5 mL sterile syringe and anticoagulated with EDTA. Genomic DNA was extracted from peripheral blood using a universal genomic DNA extraction kit according to the instructions.

Embodiment 3

[0024] Embodiment 3: detection primer and detection probe specificity test

[0025] Real-time fluorescence quantitative PCR reaction system 20 μL, including: template DNA 5 μL, 10×TaqMan buffer 2 μL, 5mM MgCl 2 1.5 μL, 2.5 U / μL HotStart DNA polymerase 1 μL, 2.5 mmol / L dNTPs 1 μL, 20 μmol / L SRV virus probe 0.25 μL, 20 μmol / L SRV-F and SRV-R each 0.25 μL, sterilized ultrapure Water 8.75 μL. The reaction program was 95°C for 30s; 95°C for 5s, 61°C for 31s, 50 cycles.

[0026] Specific test:

[0027] Genomic DNA was extracted from foamy virus, SIV, SRV-1, SRV-2, SRV-3 and SRV / D-T positive samples and peripheral blood of normal cynomolgus monkeys. Real-time fluorescent quantitative PCR amplification was performed 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 result...

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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

Technical field: [0001] The invention belongs to the technical field of bioengineering, in particular to primers, probes, detection kits and detection methods for monkey SRV virus real-time fluorescent quantitative PCR detection. Background technique: [0002] Cynomolgus monkeys and rhesus monkeys are widely used in pathology, experimental zoology and pharmacology research. During the research process, the experimental monkeys carry D-type retrovirus (simian type D retrovirus, SRV) will seriously interfere with the experimental results, and 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), immunofluorescence and so on. These methods have their limitations in terms of specificity, sensitivity, and early diagnosis. In previous serological diagnosis, according to related literature, only 1% to 4% of SRV positive serological tests were de...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
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