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Fluorescent RT-RAA (Reverse Transcription Recombinase Aid Amplification) primer for detecting MERS-CoV (Middle East Respiratory Syndrome Coronavirus), probe and detecting method

A detection method and probe technology, applied in microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems such as complex primers, achieve strong specificity, meet the needs of rapid epidemic diagnosis and whole-process monitoring, The effect of reducing the fatality rate

Inactive Publication Date: 2018-01-09
宁波国际旅行卫生保健中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the established MERS-CoV detection methods at home and abroad include real-time RT-PCR detection method, semi-nested PCR detection method, LAMP detection method, etc., but the PCR method requires the use of complex instruments and well-equipped laboratories, while the LAMP method The primers are complex and must be designed with special software, and the amplification products obtained by the LAMP method are fragments of various sizes, which cannot be directly cloned and sequenced, and can only be used to determine the existence of the target gene

Method used

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  • Fluorescent RT-RAA (Reverse Transcription Recombinase Aid Amplification) primer for detecting MERS-CoV (Middle East Respiratory Syndrome Coronavirus), probe and detecting method
  • Fluorescent RT-RAA (Reverse Transcription Recombinase Aid Amplification) primer for detecting MERS-CoV (Middle East Respiratory Syndrome Coronavirus), probe and detecting method
  • Fluorescent RT-RAA (Reverse Transcription Recombinase Aid Amplification) primer for detecting MERS-CoV (Middle East Respiratory Syndrome Coronavirus), probe and detecting method

Examples

Experimental program
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Effect test

Embodiment 1

[0053] Embodiment 1, be used to detect the design of the fluorescent RT-RAA primer of Middle East respiratory syndrome coronavirus, probe

[0054] Aiming at the specific conserved region of MERS-CoV as the target region, specific primers and fluorescent RAA probes were designed, the sequences of which are respectively:

[0055] Forward primer, such as the nucleotide sequence shown in SEQ ID No. 1 in the sequence listing:

[0056] 5'-TCCACATTGAGGGGACTGGAGGCAATAGTC-3'

[0057] Reverse primer, such as the nucleotide sequence shown in SEQ ID No. 2 in the sequence listing:

[0058] 5'-GATCACCTCCTACTGCTCCGATTCCAGATG-3'

[0059] Oligonucleotide probe, such as the nucleotide sequence shown in SEQ ID No. 3 in the sequence listing:

[0060] 5'-GAGGCAATAGTCAAATCATCTTCAAGAGCC(FAM-dT)(THF)(BHQ-dT)AGCGTAAGCAGAAAC-3';

[0061] in:

[0062] FAM-dT represents a thymidine deoxynucleotide carrying a fluorescein group FAM (6-Carboxyfluorescein);

[0063] THF represents a tetrahydrofuran (te...

Embodiment 2

[0065] Embodiment 2, the establishment of a kind of fluorescent RT-RAA method for detecting MERS-CoV

[0066] (1) Fluorescence RT-RAA reaction

[0067] 1) Extraction of sample RNA: The sample RNA can be extracted by using the viral RNA nucleic acid extraction kit from Kaiser Bioengineering Co., Ltd., according to the instructions of the kit.

[0068] 2) Using the forward primer, reverse primer and probe designed in Example 1, and the RAA reaction kit (this example specifically uses the RAA basic kit of Jiangsu Qitian Gene Biotechnology Co., Ltd.), with this example Step 1) The prepared sample RNA to be detected is used as a template to perform an amplification reaction, wherein the reaction system is as follows:

[0069] 25 μl RAA reaction buffer (provided by the RAA basic kit of Jiangsu Qitian Gene Biotechnology Co., Ltd.);

[0070] 2 μL each of the forward primer and reverse primer (10 μM) designed in Example 1;

[0071] 0.5 μL of the probe designed in Example 1 (10 μM); ...

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Abstract

The invention provides a fluorescent RT-RAA (Reverse Transcription Recombinase Aid Amplification) primer for detecting MERS-CoV (Middle East Respiratory Syndrome Coronavirus), a fluorescent probe andapplication of the fluorescent RT-RAA primer and the fluorescent probe in detecting the MERS-CoV. The fluorescent RT-RAA primer and the fluorescent probe, disclosed by the invention, are capable of quickly and qualitatively detecting the MERS-CoV in a throat swab sample, detection can be carried out under the normal temperature of 39 DEG C, a diagnosis result can be obtained within 20 minutes, andthe detection time is greatly shortened; the fluorescent RT-RAA primer and the fluorescent probe are stronger in specificity and higher in sensitivity, quick diagnosis and whole-process monitoring onan epidemic situation can be completely met, and time is bought for early diagnosing and early treating the epidemic situation, reducing a case fatality rate and controlling the epidemic situation.

Description

technical field [0001] The invention belongs to the field of in vitro nucleic acid detection, in particular to a fluorescent RT-RAA primer, probe and detection method for detecting Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in throat swab samples. Background technique [0002] Middle East Respiratory Syndrome (MERS) is a severe respiratory disease caused by Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Respiratory syndrome coronavirus (SARS-CoV) is a newly discovered coronavirus with strong transmissibility, high case fatality rate, and ability to infect humans. Since it was first discovered in Saudi Arabia in 2012, the number of infection cases has continued to increase. According to the latest statistics from the World Health Organization (WHO), as of July 1, 2017, 2,040 cases of MERS-CoV virus infection have been confirmed in the laboratory, of which 722 died , the case fatality rate reaches 35%, which is much higher than that of SARS-CoV. The inf...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 周冬根罗洁应清界俞雪钧
Owner 宁波国际旅行卫生保健中心
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