Fluorescent RT-PCR reagent and method for detecting influenza A virus, influenza B virus and coronavirus SARS-CoV-2

A technology of influenza B virus and influenza A virus, which is applied in the field of pathogenic microorganism detection, can solve the problems of reagent failure risk, increased cost of cold chain management, reduced reagent detection performance, and inability to adapt to the application of grassroots detection institutions, so as to avoid Preserve the effects of temperature changes or repeated freezing and thawing, good tolerance, and guaranteed accuracy

Inactive Publication Date: 2021-02-26
深圳市赛格诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, conventional fluorescent RT-PCR detection reagents are all liquid reagents, which need to be stored below -20°C to maintain detection activity. Temperature rise and repeated freezing and thawing during storage, transportation and use will reduce the detection performance of the reagents
Especially during the epidemic period, transportation is blocked, cold chain transportation conditions are difficult to guarantee, the risk of reagent failure and cold chain management costs greatly increase during long-distance transportation, and it cannot adapt to the application of international long-distance transportation and grass-roots testing institutions with poor cold chain conditions.
[0006] In addition, due to the outbreak of the global epidemic, different regions entered the winter influenza season separately, which brought greater challenges to epidemic control due to the similarity of early symptoms between influenza and COVID-19

Method used

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  • Fluorescent RT-PCR reagent and method for detecting influenza A virus, influenza B virus and coronavirus SARS-CoV-2
  • Fluorescent RT-PCR reagent and method for detecting influenza A virus, influenza B virus and coronavirus SARS-CoV-2
  • Fluorescent RT-PCR reagent and method for detecting influenza A virus, influenza B virus and coronavirus SARS-CoV-2

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Embodiment 1: This embodiment provides a design scheme for detecting fluorescent RT-PCR primers and probes for influenza A virus, influenza B virus, coronavirus SARS-CoV-2 and human RNase P nucleic acid.

[0046] The present invention selects the genome information and human RNase P nucleic acid sequence information of influenza A virus, influenza B virus, coronavirus SARS-CoV-2 in the public gene sequence database. figure 1 , 2 The nucleic acid sequences shown in , 3, and 4 are respectively used as the detection target sequences of influenza A virus, influenza B virus, coronavirus SARS-CoV-2 and human RNase P. Utilize the Primer Express V3.0 software to design the following primers for amplifying the target sequence and the probe sequences for detecting the target sequence:

[0047] The sequence of the specific forward primer of influenza A virus is SEQ ID No.1, Tm=59.0°C, the sequence of the reverse primer is SEQ ID No.2, Tm=58.4°C, the sequence of the probe is SEQ I...

Embodiment 2

[0053] Example 2: This example demonstrates that the influenza A virus, influenza B virus and coronavirus SARS-CoV-2 fluorescent RT-PCR detection reagents described in the present invention are made into freeze-dried reagents by vacuum freeze-drying technology.

[0054] Prepare influenza A virus, influenza B virus, and coronavirus SARS-CoV-2 fluorescent RT-PCR liquid detection reagents according to the ingredients and contents described in Table 1, and distribute them in 200 μl PCR eight-strip tubes according to 1 part / tube Carry out vacuum freeze-drying afterward, the influenza A virus that obtains, influenza B virus and coronavirus SARS-CoV-2 lyophilized fluorescent RT-PCR detection reagent are white particles ( Figure 5 ). Add 23 μl of nuclease-free water to a reagent, shake slowly for 10 seconds, the reagent is completely dissolved, and the volume measured with a pipette is 25 μl, which shows that the volume of the reagent before reconstitution is equivalent to 2 μl.

[...

Embodiment 3

[0056] Embodiment 3: the present embodiment demonstrates influenza A virus, influenza B virus with influenza A virus of the present invention, influenza B virus and coronavirus SARS-CoV-2 lyophilized fluorescent RT-PCR detection reagent , SARS-CoV-2 and human RNase P nucleic acid positive reference products detection effect.

[0057] Perform a 10-fold serial dilution of the mixture of influenza A virus, influenza B virus, SARS-CoV-2 and human RNase P RNA positive reference substance, mix 5 μl of each concentration with 18 μl of nuclease-free water, and then add to 1 aliquot In the influenza A virus, influenza B virus and coronavirus SARS-CoV-2 lyophilized fluorescent RT-PCR detection reagent prepared according to Example 2; 23 μl of nuclease-free water was used as a negative control. A reaction mixture with a volume of 25 μl was obtained, which was dissolved by slow shaking, centrifuged, and tested on a fluorescent PCR instrument. The reaction program was set according to Tabl...

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Abstract

The invention provides a freeze-dried type multiple fluorescent RT-PCR reagent and a method for detecting and distinguishing influenza A virus, influenza B virus and coronavirus SARS-CoV-2. Primers and probes of the fluorescent RT-PCR reagent for detecting the influenza A virus, the influenza B virus and the coronavirus SARS-CoV-2 provided by the invention are obtained by designing and screening based on the latest gene sequence information of the influenza A virus, the influenza B virus and the coronavirus SARS-CoV-2 in a public gene sequence database, and the reagent has good inclusivity, specificity and reaction performance. According to the reagent, a human RNase P nucleic acid fragment is used as a detection internal reference. In addition, the fluorescent RT-PCR detection reagent forthe influenza A virus, the influenza B virus and the coronavirus SARS-CoV-2 is prepared into a freeze-dried form capable of being stored at normal temperature through freeze-drying technology, thus avoiding the risk that the conventional liquid fluorescent PCR detection reagent is invalid due to the change of storage temperature or repeated freezing and thawing, and the transportation and management cost is reduced.

Description

technical field [0001] The invention relates to pathogenic microorganism detection technology, in particular to nucleic acid detection of influenza A virus, influenza B virus and coronavirus SARS-CoV-2. Background technique [0002] Respiratory tract infection is the most common type of disease in humans, and it is one of the main causes of morbidity and death in the population. Research shows that respiratory viruses are the most common pathogens causing respiratory diseases. Respiratory viruses include influenza virus, coronavirus, rhinovirus, respiratory syncytial virus, parainfluenza virus, human metapneumovirus, adenovirus, etc. The clinical manifestations caused by respiratory virus infection are relatively similar, mainly rhinitis, pharyngitis, laryngitis, tonsillitis, tracheitis, bronchitis, pneumonia, cough, dyspnea, fever, fatigue, etc., but the infection caused by different pathogens, the treatment Methods, efficacy and course of disease are not the same. [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2600/166C12Q2521/107C12Q2537/143C12Q2563/107C12Q2545/101
Inventor 黎绍基林镜中刘钰涵朱碧莹黄俊辉
Owner 深圳市赛格诺生物科技有限公司
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