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Real-time fluorescent RT-PCR detection method for MARV (Marburg virus)

A RT-PCR and real-time fluorescence technology, applied in the biological field, can solve problems such as increased risk, and achieve the effects of improved sensitivity, strong detection specificity, and good amplification effect

Pending Publication Date: 2019-03-22
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with the strengthening of economic cooperation between my country and Africa, frequent international trade, especially the increasing number of borders in recent years, the risk of monkey Marburg virus entering my country through experimental animals has also increased significantly

Method used

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  • Real-time fluorescent RT-PCR detection method for MARV (Marburg virus)
  • Real-time fluorescent RT-PCR detection method for MARV (Marburg virus)
  • Real-time fluorescent RT-PCR detection method for MARV (Marburg virus)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] A set of real-time fluorescent RT-PCR primers and probes for detection of monkey Marburg virus, including upstream primer MbV-F, downstream primer MbV-R and TaqMan probe MbV-P, the length of the amplified target fragment is 163bp, such as figure 1 As shown, the nucleotide sequences of each primer are as follows:

[0028] Upstream primer MbV-F: 5'- TTG GTG CGG ACC TCC AAA GT -3';

[0029] Downstream primer MbV-R: 5'- GCG CAA TTG CTG AGA GCT GA -3';

[0030] TaqMan probe MbV-P: 5'- FAM-AGC AGG CGT TGA GCA ACC TAG CCC GA- 3'.

[0031] The 5' and 3' ends of the probe were modified with fluorescent groups FAM and BHQ1, respectively.

Embodiment 2

[0033]Optimization of annealing temperature for real-time fluorescent RT-PCR detection method of monkey Marburg virus:

[0034] (1) Real-time fluorescent RT-PCR reaction system: 10×RT-PCR buffer 2 μL, 25 mmol / L MgCl 2 2 μL, 2.5 mmol / L dNTPs 1.5 μL, 10 μmol / L upstream and downstream primers 0.5 μL each, 10 μmol / L probe 0.5 μL, 5 U / μL DNA polymerase 0.5 μL, 5 U / μL reverse transcriptase 0.5 μL, 40 U / μL RNase inhibitor 0.5 μL, sample RNA to be tested 4 μL, then add 7.5 μL of DEPC water to make the total reaction volume 20.0 μL;

[0035] (2) Real-time fluorescence RT-PCR optimized reaction program: reverse transcription at 50°C for 30 min; pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 55°C-65°C (gradient temperature) for 30 s The temperature setting was repeated 3 times, a total of 40 cycles;

[0036] (3) Selection of annealing temperature: the optimization results of annealing temperature are as follows: figure 2 As shown, when the annealing ...

Embodiment 3

[0038] Utilize the detection method that the simian Marburg virus real-time fluorescent RT-PCR primer and probe optimization provided by the invention comprise the following steps:

[0039] (1) Real-time fluorescent RT-PCR reaction system configuration: 10×RT-PCR buffer 2 μL, 25 mmol / L MgCl 2 2 μL, 2.5 mmol / L dNTPs 1.5 μL, 10 μmol / L upstream and downstream primers 0.5 μL each, 10 μmol / L probe 0.5 μL, 5 U / μL DNA polymerase 0.5 μL, 5 U / μL reverse transcriptase 0.5 μL, 40 U / μL RNase inhibitor 0.5 μL, sample RNA to be tested 4 μL, then add 7.5 μL of DEPC water to make the total reaction volume 20.0 μL;

[0040] (2) Real-time fluorescent RT-PCR reaction program: reverse transcription at 50°C for 30 min; pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 61°C for 30 s, a total of 40 cycles, and single-point fluorescence detection at 61°C ;

[0041] (3) Judgment of results: the negative control has no Ct value and no amplification curve, and the positi...

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Abstract

The invention provides a group of real-time fluorescent RT-PCR specific primers and probe special for detecting MARV (Marburg virus) and a corresponding detection method established after a reaction system and a reaction program are optimized by the primers and the probe. The real-time fluorescent RT-PCR specific primers and probe and the detection method have the following advantages: (1) good stability and specificity: the primers and the probe have high specificity for detection of the MARV, have no cross reaction with other monkey viruses and have good repeatability; (2) high sensitivity:sensitivity can reach 6.04 copies / mu L; (3) simple and rapid operation: the whole reaction can be completed within 90 min. A kit can be used for detection of the MARV and differential diagnosis of theMARV with other money virus diseases and has great significance and practical application value for ensuring life and health safety of people and animals in China and preventing serious foreign diseases.

Description

technical field [0001] The invention relates to a real-time fluorescent RT-PCR detection method for monkey Marburg virus, which belongs to the field of biotechnology and is suitable for the detection of imported non-human primate monkey Marburg virus and the differential diagnosis of other related viral diseases. Background technique [0002] Monkey Marburg virus (MARV), also known as green monkey virus, is a highly pathogenic virus with a fatality rate as high as 90%. There is currently no specific drug for treatment. MARV belongs to the Filoviridae family (Filoviridae), a single-stranded negative-strand RNA virus with a genome of about 19.1 kb, which is the largest known negative-strand RNA virus. The linear genome is arranged in 3'-NP-VP35-VP40-GP-VP30-VP24-L-5'. The virus has only one serotype and is native to Africa. Virus-infected nonhuman primates and patients are the main source of infection. The virus is usually first transmitted to humans by infected non-human p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2561/101C12Q2521/107
Inventor 张体银李丹丹邱香果郑腾王武军张志灯林素洁
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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