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Multiple primer group for respiratory tract infection virus detection and construction method thereof

A virus detection and respiratory technology, applied in the field of biochemistry, can solve the problems of non-specific increase in the system, easy mutual interference, widening gap in amplification efficiency of different target sequences, etc.

Pending Publication Date: 2021-03-30
JIHUA LAB
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the one hand, different primer pairs are likely to interfere with each other, resulting in non-specific amplification or primer dimer, which eventually leads to an increase in the non-specificity of the system and a decrease in the amplification efficiency of the target nucleic acid; The detection system may preferentially amplify short fragments, resulting in a further widening gap in the amplification efficiency of different target sequences
These problems have hindered the construction of multiplex PCR system

Method used

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  • Multiple primer group for respiratory tract infection virus detection and construction method thereof
  • Multiple primer group for respiratory tract infection virus detection and construction method thereof
  • Multiple primer group for respiratory tract infection virus detection and construction method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0120] (1) Design 18 specific primer pairs for the genome-wide conserved sequences of the 17 common respiratory viruses mentioned in Table 1. Among them, two primer pairs are designed for the new coronavirus, and one primer pair is set for other pathogens. The sequence is shown in Table 3. In the scheme of this example, among the two primers corresponding to each respiratory pathogen, the 5' end of one primer is labeled with FAM fluorescein.

[0121] (2) Prepare the primer premix according to the final concentration of each primer in Table 3.

Embodiment 2

[0123] In the protocol of this embodiment, the target sequence in the sample to be tested is amplified by PCR amplification. The PCR amplification system preparation is shown in Table 4, and the reaction procedure is referred to in Table 5. Among them, the 2xTaq PCR master mix used was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., and SMART® MMLV Reverse Transcriptase was purchased from Baoriyi Biotechnology (Beijing) Co., Ltd.; the template was viral nucleic acid extract or artificially synthesized positive plasmid .

[0124] Table 4

[0125]

[0126] table 5

[0127]

[0128] The obtained PCR products can be analyzed initially by agarose gel electrophoresis; the 3730XL sequence analyzer is used for more precise analysis, and the data is analyzed using GENEMAPPER3.2. Comparison to the capillary electrophoresis profile of a standard sample confirms the presence of specific targets in multiplexed products.

[0129] 1. Agarose gel electrophoresis an...

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Abstract

The invention discloses a multiple primer group for respiratory tract infection virus detection and a construction method thereof; the sequence of the multiple primer group for respiratory tract infection virus detection is shown as SEQ ID NO.1-36, so that simultaneous amplification of 18 targets can be realized, and the multiple primer group is suitable for clinical screening of respiratory tractinfection cases and multiple respiratory tract virus composite infection cases. In addition, 17 possible infection types can be screened at the same time through single detection, so that the detection cost can be reduced, the detection time can be saved, and the kit is suitable for emergency detection and conventional detection of sudden hygienic events.

Description

technical field [0001] The invention relates to the field of biochemistry, and mainly relates to a multiple primer set and a construction method thereof for detecting respiratory infection viruses. Background technique [0002] Acute respiratory infections are common in children and adolescents, and have a higher fatality rate in groups with weaker resistance such as young children and elderly infected persons. Common pathogens of acute respiratory infections include bacteria, viruses, chlamydia, etc., and more than 80% of the infections are caused by respiratory viruses. Since entering the 21st century, the world has suffered from respiratory viruses many times, such as: bird flu, SARS, MERS, etc. The novel coronavirus (CoVID-19) that swept the world at the end of 2019 is still seriously endangering human health and life. Due to the short incubation period, complex clinical symptoms and similar epidemic characteristics of these viral infections, it has caused great diffic...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2537/143C12Q2563/107C12Q2565/125
Inventor 钟嘉俊顾桐旭褚春旭白鹏利
Owner JIHUA LAB
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