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Reference gene for respiratory tract RNA virus PCR detection and detection product thereof

An RNA virus and internal reference gene technology, applied in the field of internal reference genes and their detection products, can solve problems such as false negatives, and achieve the effect of improving the accuracy of diagnosis

Pending Publication Date: 2020-06-16
SHANGHAI SHEN LIAN BIOMEDICAL CORP
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This suggests that even if RNA is lost during the experiment (RNA is degraded, RNA is not effectively extracted, reverse transcription reaction fails), it is very possible to obtain "effective" detection of internal reference genes by amplifying the corresponding genomic DNA. results in a negative call, but may actually be a false negative

Method used

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  • Reference gene for respiratory tract RNA virus PCR detection and detection product thereof
  • Reference gene for respiratory tract RNA virus PCR detection and detection product thereof
  • Reference gene for respiratory tract RNA virus PCR detection and detection product thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Screening of candidate internal reference genes (homology analysis)

[0047] According to the ICG database (http: / / icg.big.ac.cn / index.php / Homo_sapiens) and public literature [Jo Vandesompele, 2002], a total of 29 internal reference genes commonly used in gene expression research were summarized (see Table 3 ). Referring to [Sun, Y, 2012], the number of pseudogenes of each internal reference gene was analyzed. Specifically, the mRNA sequence of each internal reference gene was obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov / nuccore / ), and after removing the tail Poly-A sequence, it was input into the human genome UCSC database (http: / / genome.ucsc.edu / ) for Blat alignment. The UCSC database is scored based on information such as sequence similarity, sequence length, gap, etc. The higher the score, the higher the similarity. A pseudogene with a score value of 200 or more and a similarity of 80% or more was defined as a pseudogene.

[0048] T...

Embodiment 2

[0052] Example 2 Candidate gene screening (expression level analysis)

[0053] Throat swabs are convenient for sampling and are one of the most commonly used samples for in vitro diagnosis of respiratory viruses. Therefore, the selected internal reference genes should be stably expressed in throat swab samples. In this example, based on the expression level, the 6 internal reference genes screened in Example 1 were further screened.

[0054] At present, there is no systematic research data on gene expression in throat swab samples. In this example, a reasonable prediction is made by analyzing the expression levels of each gene in nasopharyngeal tissue and skin tissue. By searching the Gene Expression Omnibus database and NCBIgene database, the expression levels of each candidate gene in healthy nasopharyngeal tissue and skin tissue were analyzed (https: / / www.ncbi.nlm.nih.gov / geo / and https: / / www .ncbi.nlm.nih.gov / gene / ). The results are analyzed as figure 2 As shown, 5 g...

Embodiment 3

[0055] Embodiment 3 primers, probe design and screening

[0056] The five genes (CYC1, HUWE1, TFRC, HMBS, IPO8) screened in Example 2 were used as candidate internal reference genes for further testing.

[0057] The mRNAs of five candidate genes were downloaded from genebank, and the corresponding primers and taqman probe sequences were obtained by designing independently or through literature research (see Table 4). For the convenience of optimization, 2 to 4 groups of candidate primers and probe combinations (respectively represented by numbers 1, 2, 3 and 4) are set for each candidate gene, wherein for the primer probe of HMBS, the HMBS-1 sequence is from the literature [ WiegerJ.Norde, 2008] [H Leroy, 2005], the rest are self-designed. The primer probe sequence of the control gene (RPP30) was from CDC, USA. Genomic DNA usually contains multiple interlaced exons and introns. In order to specifically detect mRNA and exclude the detection of genomic DNA, the designed and ...

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Abstract

The invention provides a reference gene for respiratory tract RNA virus PCR detection and a detection product thereof. The reference gene is at least one of CYC1, HUWE1, TFRC and IPO8 genes. The invention also designs a primer and a probe for the reference gene. Compared with an internal reference gene RNase P (RPP30) and a corresponding primer and probe used by the American CDC, the primer and probe for the internal reference gene designed and screened by the invention are highly specific, only mRNA amplification is carried out, and genome DNA amplification is not carried out; and the reference gene can be stably detected in people.

Description

technical field [0001] The invention relates to the technical field of respiratory RNA virus detection, in particular to an internal reference gene for PCR detection of respiratory RNA virus and a detection product thereof. Background technique [0002] Respiratory virus infection is one of the leading causes of death and disability worldwide. According to statistics, the influenza virus killed more than 100 million people in the last century [Eric T Beck, 2010]; among children under 5 years old, acute lower respiratory tract infection caused more than 20% of the deaths, among which pneumonia was the most serious, accounting for more than 90% [Jeanne Carr, 2010]. In addition, new respiratory viruses have appeared frequently in this century, such as severe acute respiratory syndrome virus (SARS virus, 2002), Middle East respiratory syndrome virus (MERS virus, 2012), new coronavirus (SARS-COV2 virus, 2019) , seriously endangering human life and causing heavy social and econo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/166G16B30/00Y02A50/30
Inventor 仲从浩张震聂东升王科杜祥月张艳
Owner SHANGHAI SHEN LIAN BIOMEDICAL CORP
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