Primer probe set, kit and detection method for detecting SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus) and MERS-CoV (Middle East Respiratory Syndrome Coronavirus)

A detection method, primer-probe technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of insufficient SNP recognition, easy pollution of experimental environment, false positives, etc., to reduce manpower Cost and time cost, highly conservative and specific, highly specific effect

Inactive Publication Date: 2018-05-22
北京卓诚惠生生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, LAMP needs to set up a separate reverse transcription step to detect RNA samples, and the reaction can be completed in 60-90 minutes, and the recognition degree of SNP is not enough, and the single point mutation in the primer binding region cannot be recognized by LAMP
LAMP uses constant temperature amplification at 65°C. If the GC content in the area to be detected is too low, too high, or the secondary structure is complex, the amplification effect will be poor
In addition, LAMP uses multiple sets of primers for rolling circle replication, resulting in a huge amount of product, which easily pollutes the experimental environment and produces false positive results.

Method used

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  • Primer probe set, kit and detection method for detecting SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus) and MERS-CoV (Middle East Respiratory Syndrome Coronavirus)
  • Primer probe set, kit and detection method for detecting SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus) and MERS-CoV (Middle East Respiratory Syndrome Coronavirus)
  • Primer probe set, kit and detection method for detecting SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus) and MERS-CoV (Middle East Respiratory Syndrome Coronavirus)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] This embodiment is used to illustrate SARS-CoV and MERS-CoV specific primer probe validation test.

[0058] (1) Synthesis of primers and probes

[0059] A reagent company was commissioned to synthesize the primer probes 1-2 shown in Table 2.

[0060] Table 2

[0061]

[0062] (2) Primer probe specificity evaluation

[0063] In vitro transcribed human coronaviruses HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63, and respiratory syncytial virus, influenza virus, human parainfluenza virus, adenovirus, and human metapneumovirus nucleic acids that infect the respiratory tract were selected as specific Samples are evaluated for nucleic acid extraction. 10 μL equal volumes of each nucleic acid template were mixed, and the integrated nucleic acid was used as a template for specificity assessment.

[0064] Prepare the reaction system as follows: 50 μL of the total system, add 29.5 μL of rehydration buffer and 2.5 μL of magnesium acetate solution (280 mmol / L) to a 0.2 mL T...

Embodiment 2

[0074] This embodiment is used to illustrate the matching of SARS-CoV specific primers, probes and MERS-CoV specific primers, probes.

[0075] (1) Combination of primers and probes

[0076] The primers and probes 1 and 2 in Example 1 were simultaneously added to the reaction system for evaluation and verification, mainly evaluating specificity, minimum detection limit and coverage.

[0077] (2) Specificity assessment

[0078] The specificity evaluation template, reaction system preparation, and RPA reaction conditions are the same as in Example 1.

[0079] The result is that SARS-CoV-specific primer probe 1 and MERS-CoV primer probe 2 are used in combination, and there is no cross-reaction with the template used for specificity evaluation, which has good specificity.

[0080] (3) Evaluation of the minimum detection limit

[0081] Use restriction endonucleases SpeI and PvuII to digest the standard plasmids recombined with SARS-CoV and MERS-CoV gene sequences, linearize them ...

Embodiment 3

[0111] This example is used to illustrate the detection method of the present disclosure.

[0112] A. Detection method and result judgment

[0113] (1) Extraction of viral nucleic acid: commercially available extraction reagents were used to extract in vitro transcribed RNA.

[0114] (2) The preparation of the reaction system is the same as the system preparation method in Example 1: the primer probe set is composed of primer probes 1 and 2 shown in Table 2 and internal quality control primer probes, and the internal quality control primer probes have SEQ ID The primers of the nucleotide sequences shown in NO.7-8, the probe sequence is: 5'-CGGATCCATCTCACTGCAATGTTTTGAGA(ROXdT)C(dSpacer)A(BHQ2dT)ACTTCTTCAAGGG-3'.

[0115] (3) RPA reaction: put the RPA tube into the real-time fluorescence RPA instrument, select FAM, CY5 and ROX as reporter groups, and the RPA reaction procedure is as follows: 39°C for 10s, 39°C for 10s, 39°C for 10s (collect fluorescence), 40 cycles.

[0116] ...

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Abstract

The invention discloses a primer probe set, kit and detection method for detecting SARS-CoV and MERS-CoV based on Recombinase Polymerase Amplification (RPA) detection. The primer probe set for detecting SARS-CoV and MERS-CoV based on the RPA detection comprises primers for nucleotide sequences as shown in SEQ ID NO.1-4 and probes for nucleotide sequences as shown in SEQ ID NO. 5-6. The invention further provides the kit for detecting SARS-CoV and MERS-CoV based on the RPA detection, wherein the kit comprises the primer probe set. By adopting the technical scheme, the sensitivity, specificity and simplicity for detecting SARS-CoV and MERS-CoV are obviously improved.

Description

technical field [0001] The disclosure relates to the field of biotechnology, in particular, to a primer-probe set, a kit and a detection method for detecting severe acute respiratory syndrome virus (SARS-CoV) and Middle East respiratory syndrome virus (MERS-CoV). Background technique [0002] The infectious atypical pneumonia that broke out in my country in 2003 was defined by the World Health Organization (WHO) as severe acute respiratory syndrome (severe acute respiratory syndrome, SARS), which is a new infectious disease. Its pathogen is a mutated coronavirus-SARS coronavirus (SARS Coronavirus, SARS-CoV). Since June 2012, cases of severe acute respiratory infection have occurred in Saudi Arabia and Qatar. The pathogen belongs to the same family as SARS-CoV, with 70% DNA homology. WHO officially named it Middle East Respiratory Syndrome Coronavirus (Middle East Respiratory Syndrome, MERS-CoV). Both SARS-CoV and MERS-CoV belong to the β genus of the subfamily Coronavirida...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2521/507C12Q2522/101Y02A50/30
Inventor 王雷王月张志强
Owner 北京卓诚惠生生物科技股份有限公司
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