CN111394517A - Internal reference gene for respiratory tract RNA virus PCR detection and detection product thereof - Google Patents
Internal reference gene for respiratory tract RNA virus PCR detection and detection product thereof Download PDFInfo
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Abstract
Compared with the internal reference gene RNase P (RPP30) for the U.S. CDC and the corresponding primer and probe, the primer and probe designed and screened by the invention aiming at the internal reference gene are highly specific, only amplify mRNA but not amplify genome DNA, and the internal reference gene can be stably detected in the crowd.
Description
Technical Field
The invention relates to the technical field of respiratory RNA virus detection, in particular to an internal reference gene for respiratory RNA virus PCR detection and a detection product thereof.
Background
Respiratory virus (respiratory virus) infection is one of the leading causes of death and disability worldwide. Statistically, the death of influenza virus in the last century is more than 1 hundred million people [ Eric T Beck,2010 ]; in children under 5 years of age, acute lower respiratory tract infections account for over 20%, with pneumonia being the most severe, accounting for over 90% [ JeanneCarr,2010 ]. In addition, new respiratory viruses frequently appear in this century, such as severe acute respiratory syndrome virus (SARS virus, 2002), middle east respiratory syndrome virus (MERS virus, 2012), and new coronavirus (SARS-COV2 virus, 2019), which seriously endanger human life and cause disastrous socio-economic losses.
The method mainly comprises the steps of reverse transcription-real-time fluorescence quantitative PCR (RT-qPCR), reverse transcription-common PCR, reverse transcription-loop-mediated isothermal amplification (RT-L AMP), reverse transcription-amplification of nucleic acid sequences (RT-NASBA), reverse transcription-rolling circle amplification (RT-RCA), reverse transcription-single primer isothermal amplification (RT-SPIA), reverse transcription-dependent helicase DNA isothermal amplification (RT-HDA), reverse transcription-recombinase polymerase amplification (RT-RPA), reverse transcription-strand displacement amplification (RT-SDA) and other gene amplification technologies.
TABLE 1 respiratory viruses and corresponding viral nucleic acids [ Somerville, L. K.2015]
The attached notes of the table are as follows: ss is single-stranded, ds is double-stranded, (+) is positive (single-stranded viral RNA can function as mRNA directly), (-) is negative (single-stranded viral RNA must first synthesize complementary strand, which functions as mRNA)
Despite its many advantages, false negatives remain one of the major problems with current PCR assays, particularly RT-qPCR. The reason for false negative RT-qPCR results for newly discovered viruses may come from several aspects: 1) the pathological characteristics of the newly found virus are not completely clear, and the proper pathological sample collecting position and collecting time cannot be accurately judged; 2) the newly developed diagnostic kits have uneven detection sensitivity; 3) RNA stability is far lower than that of DNA, and related diagnostic experiments involve a plurality of processes including clinical sample collection, sample storage and transportation, RNA extraction and RT-qPCR reaction, wherein the RNA degradation or the failure of amplification reaction caused by any one-step misoperation can cause the false negative of the experiment.
In fact, the CDC in the United states applies the P30 gene (RPP30) of the reference gene RNase P to the pathogen RT-qPCR diagnosis [ Shannon L. Emery, 2004] to improve the reliability of the diagnosis result as early as in the SARS period, and the gene and related primer probe sequences are used so far (see sequence Table 2). In China, the quality control method generally adopted at present is that after the sample is transported to a laboratory, an exogenous gene or a pseudogene containing the pseudogene is added before the nucleic acid extraction or before the RT-qPCR, and the method cannot effectively monitor the upstream collection and transportation of the exogenous sample, and the experimental operation steps and the additional transportation cost are increased.
TABLE 2 primers and probes for reference Gene RNase P
Further analysis of the amplified sequence against the reference gene RPP30 revealed that it was 100% identical in human genomic DNA (fig. 1), i.e. the corresponding primers and probes both amplified RNA and amplified genomic DNA. This suggests that even if RNA is lost during the experiment (RNA is degraded, RNA is not extracted efficiently, reverse transcription reaction fails), it is likely that "efficient" detection of the reference gene is obtained by amplification of the corresponding genomic DNA, resulting in negative determination, but may actually be false negative. Indeed, the american CDC has also suggested that primers and probes directed against RPP30 can amplify both RNA and DNA simultaneously and are not suitable as quality control for reverse transcription [ US CDC, RT-PCR for Mumps Virus RNA,2010 ].
Disclosure of Invention
The invention provides an internal reference gene for respiratory RNA virus PCR detection and a detection product thereof based on the defects of the prior art, the invention screens a new internal reference gene PO L R2A and related primer and probe sequences for monitoring the experimental process of respiratory RNA virus RT-qPCR, the gene is usually used as the normalization of tumor gene expression quantification in the past, and the gene is screened out in the invention and is firstly applied to the quality control of respiratory RNA virus PCR diagnosis.
The purpose of the invention is realized by the following technical scheme:
in a first aspect, the invention provides an internal reference gene for PCR detection of respiratory RNA virus, wherein the internal reference gene is PO L R2A gene.
Preferably, the respiratory RNA virus comprises any one of severe acute respiratory syndrome virus, middle east respiratory syndrome virus, novel coronavirus, influenza virus, Respiratory Syncytial Virus (RSV), parainfluenza virus (PIV), human metapneumovirus (hMPV), Human Rhinovirus (HRV), Human Enterovirus (HEV). The nasopharyngeal swab is one of the most common and universal clinical samples for PCR diagnosis of the respiratory RNA viruses, and the internal reference gene TBP, the primer and the probe combination which are suitable for the pharyngeal swab sample are obtained by screening, so that the gene, the primer and the probe combination can be applied to the PCR diagnosis method of the respiratory RNA viruses containing any one of the viruses to improve the diagnosis accuracy.
Preferably, the PCR detection includes any one of gene amplification techniques such as reverse transcription-real-time fluorescent quantitative PCR (RT-qPCR) detection, reverse transcription-general PCR detection, reverse transcription-loop-mediated isothermal amplification (RT-L AMP) detection, reverse transcription-nucleic acid sequence-dependent amplification (RT-NASBA) detection, reverse transcription-rolling circle amplification (RT-RCA) detection, reverse transcription-single primer isothermal amplification (RT-SPIA) detection, reverse transcription-dependent helicase DNA isothermal amplification (RT-HDA), reverse transcription-recombinase polymerase amplification (RT-RPA), reverse transcription-strand displacement amplification (RT-SDA) and the like, which are all used for amplifying the number of gene sequences to be detected by designing corresponding primers to promote the gene to be detected, so that the reference gene PO L R2A screened by the present invention can be applied to any one of the PCR detection methods described above to improve the diagnosis accuracy.
In a second aspect, the invention provides a primer for detecting the aforementioned reference gene, wherein the primer sequence of the PO L R2A gene is shown as SEQ ID No.37 and SEQ ID No. 38.
In a third aspect, the invention provides a probe for detecting the reference gene as described above, wherein the probe sequence of the PO L R2A gene is shown as SEQ ID NO. 39.
In a fourth aspect, the invention provides a novel coronavirus RT-PCR detection product, which comprises a primer and a probe for detecting the gene PO L R2A of the internal reference gene, wherein the primer sequence of the gene PO L R2A is shown as SEQ ID NO.37 and SEQ ID NO.38, and the probe sequence is shown as SEQ ID NO. 39.
Preferably, the detection product comprises a detection kit and a detection test paper.
In a fifth aspect, the present invention provides a method for screening an internal reference gene for respiratory RNA virus PCR detection, comprising the following steps:
A. carrying out homology analysis on related genes, and screening genes with the pseudogene factor of 1 or less as candidate genes;
B. analyzing the expression level of each candidate gene in healthy nasopharyngeal and skin tissues to infer the expression level thereof in a nasopharyngeal swab sample, and selecting a candidate gene expressed in at least one tissue;
C. and (3) carrying out expression stability analysis on the candidate gene, and screening to obtain a gene which can be stably detected in the population, namely the reference gene.
Compared with the prior art, the invention has the following beneficial effects:
compared with the existing reference gene RPP30, the primer and the probe of the new reference gene are highly specific, only mRNA is recognized, and no cross reaction with human genome DNA is caused, in addition, the new reference gene can be stably detected in the population, the integrity of virus RNA in a clinical throat swab sample and the correctness of an experimental process can be more truly monitored by using the reference gene, the corresponding primer and the corresponding probe, and the PCR diagnosis accuracy of the respiratory RNA virus is improved.
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Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a diagram of a comparison of a human chromosome 10 DNA sequence and a sequence to be amplified of a conventional reference gene RPP 30;
FIG. 2 is a graph showing the expression levels (mean. + -. SD) of the respective genes in healthy nasopharyngeal tissues and skin tissues in example 2;
FIG. 3 shows Ct values obtained by RT-qPCR and qPCR of candidate reference genes in 12 throat swab samples;
FIG. 4 shows the results of analysis of gene expression stability.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit of the invention. All falling within the scope of the present invention.
Example 1 candidate reference Gene screening (homology analysis)
A total of 33 reference genes commonly used in gene expression studies were summarized based on the ICG database (http:// ICG. big. ac. cn/index. php/Homo _ sapiens) and published literature data [ Jo Vandesoppele, 2002] (see Table 3). Reference [ Sun, Y,2012], analyzes the pseudogene factor of each reference gene. Specifically, the mRNA sequence of each reference gene is obtained from an NCBI database (https:// www.ncbi.nlm.nih.gov/nuccore /), and after a tail Poly-A sequence is removed, the mRNA sequence is input into a 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 and the like, and the higher the score is, the higher the similarity is represented. Pseudogenes were defined as those having a score of 200 or more and a degree of similarity of 80% or more.
The analysis results are shown in Table 3, and out of 33 common reference genes, 26 genes had pseudogenes and 7 genes had no pseudogenes. The small number of pseudogenes represents that the difficulty of designing specific primers and probes is reduced, and the possibility of cross reaction with genome DNA is reduced. Based on this consideration, this example selects 9 genes with a pseudogene number of 1 or less for further screening.
TABLE 3 multiple reference genes homologous pseudogenes present in the human genome
Example 2 candidate Gene screening (expression level analysis)
The pharyngeal swab is convenient to sample and is one of the most commonly used samples for in vitro diagnosis of the respiratory viruses, so the selected internal reference gene can be stably expressed in the pharyngeal swab sample. This example further screens the 9 reference genes screened in example 1 based on expression levels.
The results of Gene Expression analysis in healthy nasopharyngeal and skin tissues (https:// www.ncbi.nlm.nih.gov/geo/and https:// www.ncbi.nlm.nih.gov/Gene /) are shown in FIG. 2, 9 genes (DDX5, PO L R2A, CYC1, SF3A1, WE1, TFRC, HMBS, TBP, HUIPO 8) are obviously expressed in the nasopharyngeal or skin tissues, thereby preliminarily predicting that the genes are also highly likely to be expressed in the pharyngeal swab sample and can be used as internal reference genes of the pharyngeal swab sample.
Example 3 primer, Probe design and screening
The 9 genes (DDX5, PO L R2A, CYC1, SF3a1, HUWE1, TFRC, HMBS, TBP, IPO8) obtained by screening in example 2 were used as candidate reference genes for further testing.
Downloading mRNA of 9 candidate genes from genebank, and obtaining corresponding primer and taqman probe sequences (see Table 4) by self-design or literature research, for convenience of preference, 2 to 4 sets of candidate primer and probe combinations (numbered 1, 2, 3 and 4, respectively) were set for each candidate gene, wherein for primer probes for HMBS and TBP, the HMBS-1 and TBP-2 sequences were from the literature [ Wieger J. Norde,2008] [ H L eroy, 2005], the remainder were self-designed, the primer probe sequence for the control gene (RPP30) was from U.S. CDC.
According to the detection curve of the nucleic acid sample of the pharyngeal swab, the optimal primer probe combination aiming at 9 internal reference genes is preliminarily screened, and particularly, the pharyngeal swab of a healthy person is collected and used for obtaining Pure L inkTMNucleic acid was extracted from Viral RNA/DNA Mini Kit (ABI) using TaqManTMThe amplification effect of each primer and probe was examined and compared separately by Fast Virus 1-Step Master Mix (ABI), and the specific experimental method was referred to in example 4.
The method specifically comprises the steps of optimizing the annealing temperature of each primer probe combination, detecting the amplification efficiency of each primer probe combination through a gradient dilution sample, analyzing the amplification specificity through blast and nucleic acid electrophoresis, selecting a specific primer probe combination with the amplification efficiency of 0.8-1.2 for further analysis, and specifically obtaining the optimized primer probe combinations of HMBS-1, TBP-3, SF3A1-2, PO L R2A-2, DDX5-2, CYC1-1, HUWE1-1, TFRC-2 and IP08-1, wherein the optimal annealing temperature/amplification efficiency of each primer probe combination is respectively 60 ℃/0.83, 64 ℃/0.82, 64 ℃/0.97, 60 ℃/1.12, 56 ℃/0.91, 58 ℃/0.87, 58 ℃/0.89, 58 ℃/0.9 and 60 ℃/1.1.
TABLE 4 primer and taqman Probe sequence information
Example 4 validation of the specificity and stability of detection of candidate reference genes in clinical samples
1. Throat collecting swab
Pharyngeal swab samples of 12 healthy persons (8 women and 4 men) were collected, specifically, medical disposable sterile cotton swabs were gently scraped 30 times on the left and right sides of the pharyngeal portion of the subject, the cotton swabs were placed in a 1.5ml centrifuge tube, and 500 μ L DEPC water was added to the tube.
2. Extraction of RNA from throat swab
The swabs collected were numbered and immediately thereafter Pure L ink was usedTMViral RNA/DNA Mini Kit (ABI) extracts nucleic acids, specifically, 25 μ L proteinase K, 200 μ L throat swab sample, 200 μ L lysate (containing 5.6 μ g of carrier RNA) are sequentially added to a 1.5ml sterile RNase-free centrifuge tube, a centrifuge tube cover is covered, the tube wall is slightly reversed for 15sec, incubation is performed at 56 ℃ for 15min, a brief centrifugation is performed to make the liquid on the tube wall fall to the tube bottom, 250 μ L absolute ethyl alcohol (the final concentration of ethyl alcohol is 37%) is added, the centrifuge tube cover is covered, 15sec is slightly reversed, incubation is performed at room temperature for 5min, a brief centrifugation is performed to make the liquid on the tube wall fall to the tube bottom, the lysate is transferred to an adsorption column, the centrifuge tube cover is covered, 6800g is centrifuged for 1min, the permeate is discarded, the adsorption column is placed in a wash tube (2m L), 500 μ L wash solution (WIqI) is added, the centrifuge tube cover is covered, 6800g is placed in a wash tube (2m L min), the wash tube is removed, the wash tube is placed in a wash tube (12000 g wash tube) again, the wash tube is placed in a wash tube (3500 g wash tube) with wash tube (L g wash tube) for one time, the wash tube is added, the wash tube.
RT-qPCR and qPCR detection
Each set of preferred primers and probes (see results in example 3), TaqManTMThe Fast Virus 1-StepMaster Mix (ABI) and throat swab nucleic acid samples were prepared according to Table 5, with a final reaction volume of 20. mu. L, final primer and probe concentrations of 500nM and 200 nM., respectively, by RT-qPCR on an ABI7500Fast quantitative fluorescence PCR instrument, using 1 cycle (50 ℃, 5min), 1 cycle (95 ℃, 30sec), 40 cycles (95 ℃, 15 sec; 60 ℃, 60sec), collecting Cy5 fluorescence at 60 ℃ extension phase, RPP30 as control (see Table 4), sterile RNase-free water as negative control, three replicates per set, based on amplification curve, and using the sameThe line automatically calculates the Ct value.
TABLE 5 PCR reaction System preparation
To verify the specificity of RNA detection, qPCR is further carried out, the Taq enzyme used in qPCR only can use DNA as a template and cannot use RNA as a template, and mix reaction solution (Premix Ex Taq) is selectedTMmix (takara)) contains thermostable RnaseH and can further inhibit the amplification of RNA, so whether or not it cross-reacts with genomic DNA can be preliminarily determined based on the presence or absence of qPCR amplification signal. The method comprises the following steps: each primer and probe set, Premix Ex TaqTMmix (takara), and throat swab nucleic acid samples were prepared as per Table 5, with a final reaction volume of 20. mu. L, final primer and probe concentrations of 500nM and 200 nM., respectively, by RT-qPCR on an ABI7500fast fluorescence quantitative PCR instrument using 1 cycle (95 ℃,20 sec), 40 cycles (95 ℃, 5 sec; 60 ℃,20 sec), Cy5 fluorescence collected during the 60 ℃ extension phase, RPP30 as a control, sterile RNase-free water as a negative control, and Ct values were automatically calculated from the amplification curves for each set of three replicates.
The detection result is shown in FIG. 3, the designed related primer probes have amplification signals only in RT-qPCR reaction but not in qPCR reaction for 9 candidate genes (DDX5, PO L R2A, CYC1, SF3A1, HUWE1, TFRC, HMBS, TBP and IPO8) and have amplification signals in RT-qPCR and qPCR for the control gene RPP30, and the result shows that only mRNA but not genomic DNA is specifically detected for the primer probe combination of each internal reference gene, and the mRNA and genomic DNA can be simultaneously detected for the primer probe combination of the control internal reference gene RPP30, which is poor in specificity.
In the aspect of detection rate, except for the low detection rate of HMBS (only 42%), the detection rate of all the other candidate reference genes is 100%, which is consistent with the detection rate of the reference gene RPP 30. The detection rate of the HMBS gene is low, probably because the gene is expressed in a throat swab sample at a low level, and the detection result is unstable.
TABLE 6 detection rates of various candidate reference genes in 12 throat swab samples
The results of the above studies preliminarily confirm that the prediction model based on the analysis of homologous pseudogenes and tissue expression level used in the present study is basically established for the reference gene. Eliminating genome DNA homologous interference through homology analysis can reduce the difficulty of designing specific primers and probes and ensure the specific detection of mRNA; the mRNA level of the gene in the tissue can be preliminarily estimated from the expression level.
4. Analysis of Gene expression stability
The higher the stability of gene expression, the more stably detected in the population and the higher the reliability for diagnosis. The expression stability of the candidate reference gene was analyzed by RefFinder online software and compared with the control gene RPP 30. RefFinder integrates the functions of the most mainstream algorithms (geNorm, Normfinder, BestKeeper and Delta-Ct method) at present, carries out comprehensive scoring [ F Xie,2012], and low score represents strong gene expression stability.
The results are shown in fig. 4, among the 9 candidate reference genes, the scores of PO L R2A, TBP and SF3a1 are significantly lower than that of the control gene RPP30, and the scores of DDX5, TFRC, CYC1, IPO8 and HUWE1 are closer to that of the control gene RPP30, while the score of HMBS is much higher than that of RPP 30.
Therefore, the PO L R2A with the highest expression stability is used as an internal reference gene in subsequent experiments.
Example 5 PO L R2A as reference gene detection applied to novel coronavirus RT-qPCR diagnosis
In this embodiment, the detection of the PO L R2A reference gene is integrated into the existing novel coronavirus RT-qPCR diagnostic method, and the specific operations are as follows:
the primers and probes for the novel coronavirus ORF1ab gene and N gene are disclosed by the Chinese disease prevention and control center and synthesized by the Biotechnology engineering (Shanghai) GmbH, and the specific sequences are shown in Table 7.
TABLE 7 novel coronavirus RT-qPCR detection primer and Probe information
Referring to the experimental method of example 4, throat swabs of 5 healthy persons were collected, nucleic acids were extracted, and viral genes (1ab and N) and reference gene (PO L R2A) were detected by RT-qPCR, respectively, RNA of the fragment to be amplified for positive control (synthesized by takara corporation), and sterile RNase-free water for negative control, before the results were judged, it was judged whether the experiment was established or not, and the establishment criteria of the experiment were that there was no Ct value for negative control, Ct value for positive control was <37, and Ct value for reference gene PO L R2A was < 37.
The result judgment refers to the standard of Chinese disease prevention and control center:
(1) negative, no Ct value or 40 Ct.
(2) Positive: ct values <37, may be reported as positive.
(3) And (3) suspicious: and (4) recommending repeated experiments when the Ct value is between 37 and 40, if the Ct value of the redo result is less than 40, the amplification curve has obvious peaks, and the sample is judged to be positive, otherwise, the sample is negative.
As shown in Table 8, 5 throat swab samples were obtained in which none of the viral genes (1ab and N) was detected, while the internal reference gene PO L R2A was stably detected, the mRNA of PO L R2A was constantly detected, indicating that the whole procedure, the reagents used and all reactions were correct, the novel coronavirus RNA was not detected, and the result was true negative.
TABLE 85 healthy human pharyngeal swab samples New coronavirus RT-qPCR results (Ct values)
| |
|
Sample 3 | Sample No. 4 | Sample No. 5 | |
| POLR2A | 28.7 | 29.2 | 29.3 | 28.5 | 30.2 |
| 1ab | Ct-free | Ct-free | Ct-free | Ct-free | Ct-free |
| N | Ct-free | Ct-free | Ct-free | Ct-free | Ct-free |
Example 6 PO L R2A as reference Gene detection for MERS Virus RT-qPCR diagnosis
In this embodiment, the PO L R2A reference gene detection is integrated into the existing Mers virus RT-qPCR diagnostic method, and the specific operations are as follows:
primers and probes for the Mers virus upE gene, the N2 gene and the N3 gene were published by the American centers for disease prevention and control and synthesized by Biotechnology engineering (Shanghai) GmbH, and the specific sequences are shown in Table 9.
TABLE 9 Mers Virus RT-qPCR detection primer and Probe information
Referring to the experimental method of example 4, throat swabs of 5 healthy persons were collected, nucleic acids were extracted, and viral genes (upE gene, N2 gene, N3 gene) and reference gene (PO L R2A) were detected by RT-qPCR, respectively, RNA of a fragment to be amplified for positive control (manufactured by takara corporation), sterile RNase-free water for negative control, and whether the experiment was established or not was judged before the result was judged.
The experimental establishment standards comprise that the negative control has no Ct value, the positive control detects the Ct value less than 37, and the internal reference gene PO L R2A detects the Ct value less than 37.
Results determination reference the criteria of the U.S. CDC:
(1) negative, no Ct value.
(2) Positive: sigmoidal amplification curve with Ct value < 40.
As shown in Table 10, in 5 throat swab samples, none of the viral genes (upE gene, N2 gene, N3 gene) was detected, while the internal reference gene PO L R2A was stably detected, and the Ct value was in the effective range, the constant detection of mRNA of PO L R2A indicated that the whole procedure, the reagents used and all reactions were correct, the Mers virus RNA was not detected, and the result was true negative.
TABLE 105 healthy human pharyngeal swab samples Mers RT-qPCR results (Ct values)
| |
|
Sample 3 | Sample No. 4 | Sample No. 5 | |
| POLR2A | 29.0 | 29.1 | 29.5 | 30.8 | 29.7 |
| upE gene | Ct-free | Ct-free | Ct-free | Ct-free | Ct-free |
| N2 gene | Ct-free | Ct-free | Ct-free | Ct-free | Ct-free |
| N3 gene | Ct-free | Ct-free | Ct-free | Ct-free | Ct-free |
Example 7 PO L R2A as reference Gene assay for SARS Virus RT-qPCR diagnosis
In this embodiment, the PO L R2A reference gene detection is integrated into the existing SARS virus RT-qPCR diagnostic method, and the specific operations are as follows:
primers and probes for the Sars virus N3 gene and M gene were published by the American centers for disease prevention and control and synthesized by Biotechnology engineering (Shanghai) GmbH, and the specific sequences are shown in Table 11.
TABLE 11 Sars Virus RT-qPCR detection primers and Probe information
Referring to the experimental method of example 4, throat swabs of 5 healthy persons were collected, nucleic acids were extracted, and viral genes (N3 gene and M gene) and reference gene (PO L R2A) were detected by RT-qPCR, respectively, RNA of a fragment to be amplified for positive control (manufactured by takara Co., Ltd.) and sterile RNase-free water for negative control were used, and before the results were judged, it was judged whether the experiment was established.
The experimental establishment standards comprise that the negative control has no Ct value, the positive control detects the Ct value less than 37, and the internal reference gene PO L R2A detects the Ct value less than 37.
The results were judged with reference to the american centers for disease prevention and control criteria:
(1) negative, no Ct value.
(2) Positive: sigmoidal amplification curve with Ct value < 40.
As shown in Table 12, in 5 throat swab samples, no viral genes (N3 gene and M gene) were detected, while the internal reference gene PO L R2A was stably detected, and the Ct value was in the effective range, the constant detection of mRNA of PO L R2A indicated that the entire operation, the reagents used and all reactions were correct, and Sars viral RNA was not detected, which was true negative.
TABLE 125 healthy human pharyngeal swab samples Sars RT-qPCR results (Ct values)
Example 8 PO L R2A as reference gene detection applied to influenza virus RT-qPCR diagnosis
In this embodiment, the detection of the PO L R2A reference gene is integrated into the influenza virus RT-qPCR diagnostic method, and the specific operations are as follows:
the universal detection primers and probes for influenza virus are disclosed by the American centers for disease prevention and control and synthesized by Biotechnology engineering (Shanghai) GmbH, and the specific sequences are shown in Table 13.
TABLE 13 influenza RT-qPCR detection primer and Probe information
Referring to the experimental method of example 4, throat swabs of 5 healthy persons were collected, nucleic acids were extracted, influenza virus genes and reference genes (PO L R2A) were detected by RT-qPCR, respectively, RNA of a fragment to be amplified for positive control (manufactured by Takara Co., Ltd.) and sterile RNase-free water for negative control were used, and before the results were judged, it was judged whether the experiment was established.
The experimental establishment standards comprise that the negative control has no Ct value, the positive control detects the Ct value less than 37, and the internal reference gene PO L R2A detects the Ct value less than 37.
And (4) result judgment standard:
(1) negative, no Ct value.
(2) Positive: sigmoidal amplification curve with Ct value < 40.
As shown in Table 14, the influenza virus genes were not detected in all of the 5 throat swab samples, while the internal reference gene PO L R2A was stably detected, and the Ct value was within the effective range, the mRNA of PO L R2A was constantly detected, indicating that the whole procedure, the reagents used and all reactions were correct, the influenza RNA was not detected, and the result was true negative.
TABLE 145 healthy human pharyngeal swab samples influenza virus RT-qPCR results (Ct values)
| |
|
Sample 3 | Sample No. 4 | Sample No. 5 | |
| POLR2A | 30.2 | 30.3 | 31.5 | 29.7 | 28.6 |
| Influenza virus gene | Ct-free | Ct-free | Ct-free | Ct-free | Ct-free |
Example 9 PO L R2A as reference Gene detection for application in respiratory syncytial Virus RT-qPCR diagnostics
In the embodiment, the detection of the PO L R2A reference gene is integrated into the prior RT-qPCR diagnostic method for the respiratory syncytial virus, and the specific operations are as follows:
primers and probes for respiratory syncytial virus genes were published by the U.S. centers for disease prevention and control and synthesized by Biotechnology engineering (Shanghai) GmbH, and the specific sequences are shown in Table 15.
TABLE 15 respiratory syncytial virus RT-qPCR detection primers and Probe information
Referring to the experimental method of example 4, throat swabs of 5 healthy persons were collected, nucleic acids were extracted, and respiratory syncytial virus gene and reference gene (PO L R2A) were detected by RT-qPCR, respectively, RNA of a fragment to be amplified for positive control (manufactured by takara Co., Ltd.) and sterile RNase-free water for negative control were used, and before the results were judged, it was judged whether the experiment was established.
The experimental establishment standards comprise that the negative control has no Ct value, the positive control detects the Ct value less than 37, and the internal reference gene PO L R2A detects the Ct value less than 37.
And (4) result judgment standard:
(1) negative, no Ct value.
(2) Positive: sigmoidal amplification curve with Ct value < 40.
As shown in Table 16, the respiratory syncytial virus gene was not detected, the internal reference gene PO L R2A was stably detected, and the Ct value was within the effective range in the 5 throat swab samples, the mRNA of PO L R2A was constantly detected, indicating that the whole operation, the reagents used and all reactions were correct, the respiratory syncytial virus RNA was not detected, and the result was true negative.
TABLE 165 healthy human pharyngeal swab samples respiratory syncytial virus RT-qPCR results (Ct values)
| |
|
Sample 3 | Sample No. 4 | Sample No. 5 | |
| POLR2A | 28.5 | 22.7 | 29.4 | 29.5 | 29.7 |
| F gene | Ct-free | Ct-free | Ct-free | Ct-free | Ct-free |
Example 10 PO L R2A as reference Gene assay for parainfluenza Virus RT-qPCR diagnostics
In this embodiment, the detection of the PO L R2A reference gene is integrated into the existing parainfluenza virus RT-qPCR diagnostic method, and the specific operations are as follows:
primers and probes for parainfluenza virus genes reference [ Templeton KE,2004], synthesized by Biotechnology (Shanghai) GmbH, with specific sequences as shown in Table 17.
TABLE 17 parainfluenza RT-qPCR detection primers and Probe information
Referring to the experimental method of example 4, throat swabs of 5 healthy persons were collected, nucleic acids were extracted, and viral genes and reference genes (PO L R2A) were detected by RT-qPCR, respectively.
The experimental establishment standards comprise that the negative control has no Ct value, the positive control detects the Ct value less than 37, and the internal reference gene PO L R2A detects the Ct value less than 37.
Criteria for result determination:
(1) negative, no Ct value.
(2) Positive: sigmoidal amplification curve with Ct value < 40.
As shown in Table 18, the virus gene was not detected, the internal reference gene PO L R2A was stably detected, and the Ct value was in the effective range in 5 throat swab samples, the mRNA of PO L R2A was constantly detected, indicating that the whole procedure, the reagents used and all reactions were correct, the parainfluenza RNA was not detected, and the result was true negative.
TABLE 185 samples of healthy human pharyngeal swab parainfluenza Virus RT-qPCR results (Ct values)
Example 11 PO L R2A as reference Gene detection for RT-qPCR diagnosis of human metapneumovirus
In this embodiment, the detection of the PO L R2A reference gene is integrated into the existing RT-qPCR diagnostic method for human metapneumovirus, and the specific operations are as follows:
primers and probes for human metapneumovirus genes reference (Jennifer Klemenc, 2012), synthesized by bio-engineering (shanghai) gmbh, and the specific sequences are shown in table 19.
TABLE 19 human metapneumovirus RT-qPCR detection primers and Probe information
Referring to the experimental method of example 4, throat swabs of 5 healthy persons were collected, nucleic acids were extracted, and the human metapneumovirus gene and the reference gene (PO L R2A) were detected by RT-qPCR, respectively.
The experimental establishment standards comprise that the negative control has no Ct value, the positive control detects the Ct value less than 37, and the internal reference gene PO L R2A detects the Ct value less than 37.
Result determination reference standard:
(1) negative, no Ct value.
(2) Positive: sigmoidal amplification curve with Ct value < 40.
As shown in Table 20, the virus gene was not detected, the internal reference gene PO L R2A was stably detected, and the Ct value was in the effective range in 5 throat swab samples, the mRNA of PO L R2A was constantly detected, indicating that the whole procedure, the reagents used and all reactions were correct, the human metapneumovirus RNA was not detected, and the result was true negative.
TABLE 205 samples of healthy human pharyngeal swab human metapneumovirus RT-qPCR results (Ct values)
Example 12 PO L R2A as reference gene detection applied to human rhinovirus RT-qPCR diagnosis
In this embodiment, the detection of the PO L R2A reference gene is integrated into the existing human rhinovirus RT-qPCR diagnostic method, and the specific operations are as follows:
primers and probes for human rhinovirus genes reference [ Julie Dupoey, 2014] was synthesized by Biotechnology engineering (Shanghai) GmbH, and the specific sequences are shown in Table 21.
TABLE 21 human rhinovirus RT-qPCR detection primers and Probe information
Referring to the experimental method of example 4, throat swabs of 5 healthy persons were collected, nucleic acids were extracted, and viral genes and reference genes (PO L R2A) were detected by RT-qPCR, respectively.
The experimental establishment standards comprise that the negative control has no Ct value, the positive control detects the Ct value less than 37, and the internal reference gene PO L R2A detects the Ct value less than 37.
Result determination reference standard:
(1) negative, no Ct value.
(2) Positive: sigmoidal amplification curve with Ct value < 40.
As shown in Table 22, the virus gene was not detected in any of the 5 throat swab samples, while the internal reference gene PO L R2A was stably detected, and the Ct value was within the effective range, the mRNA of PO L R2A was constantly detected, indicating that the whole procedure, the reagents used and all reactions were correct, the human rhinovirus RNA was not detected, and the result was true negative.
TABLE 225 samples of healthy human pharyngeal swab human rhinovirus RT-qPCR results (Ct values)
| |
|
Sample 3 | Sample No. 4 | Sample No. 5 | |
| POLR2A | 30.1 | 29.9 | 29.7 | 28.2 | 27.3 |
| Human rhinovirus gene | Ct-free | Ct-free | Ct-free | Ct-free | Ct-free |
Example 13 PO L R2A as reference gene detection applied to human enterovirus RT-qPCR diagnosis
In this embodiment, the detection of the PO L R2A reference gene is integrated into the existing human enterovirus RT-qPCR diagnostic method, and the specific operations are as follows:
primers and probes for human enterovirus genes reference [ Antonio Piralla,2015], synthesized by Biotechnology (Shanghai) GmbH, with specific sequences shown in Table 23.
TABLE 23 human enterovirus RT-qPCR detection primers and Probe information
Referring to the experimental method of example 4, throat swabs of 5 healthy persons were collected, nucleic acids were extracted, and viral genes and reference genes (PO L R2A) were detected by RT-qPCR, respectively.
The experimental establishment standards comprise that the negative control has no Ct value, the positive control detects the Ct value less than 37, and the internal reference gene PO L R2A detects the Ct value less than 37.
Result determination reference standard:
(1) negative, no Ct value.
(2) Positive: sigmoidal amplification curve with Ct value < 40.
As shown in Table 24, the constant detection of mRNA from PO L R2A indicated that the entire procedure, reagents used and all reactions were correct, that human enterovirus RNA was not detected, and that the results were true negative, while the viral genes were not detected in all of the 5 throat swab samples, the internal reference gene PO L R2A was stably detected, and the Ct value was within the effective range.
TABLE 245 healthy human pharyngeal swab samples human enterovirus RT-qPCR results (Ct values)
| |
|
Sample 3 | Sample No. 4 | Sample No. 5 | |
| POLR2A | 29.5 | 28.4 | 27.6 | 29.9 | 29.9 |
| D68 gene | Ct-free | Ct-free | Ct-free | Ct-free | Ct-free |
Example 14 PO L R2A as reference Gene detection for human coronavirus RT-qPCR diagnosis
In this embodiment, the detection of the PO L R2A reference gene is integrated into the existing human coronavirus RT-qPCR diagnostic method, and the specific operations are as follows:
primers and probes for human coronavirus genes are disclosed in the references Susan S. Chiu, 2005, center for disease prevention and control, synthesized by Biotechnology engineering (Shanghai) GmbH, and the specific sequences are shown in Table 25.
TABLE 25 human coronavirus RT-qPCR detection primers and Probe information
Referring to the experimental method of example 4, throat swabs of 5 healthy persons were collected, nucleic acids were extracted, and viral genes and reference genes (PO L R2A) were detected by RT-qPCR, respectively.
The experimental establishment standards comprise that the negative control has no Ct value, the positive control detects the Ct value less than 37, and the internal reference gene PO L R2A detects the Ct value less than 37.
Result determination reference standard:
(1) negative, no Ct value.
(2) Positive: sigmoidal amplification curve with Ct value < 40.
As shown in Table 26, the virus gene was not detected in any of the 5 throat swab samples, while the internal reference gene PO L R2A was stably detected, and the Ct value was within the effective range, the mRNA of PO L R2A was constantly detected, indicating that the whole procedure, the reagents used and all reactions were correct, the human coronavirus RNA was not detected, and the result was true negative.
TABLE 265 samples of healthy human pharyngeal swab human coronavirus RT-qPCR results (Ct values)
| |
|
Sample 3 | Sample No. 4 | Sample No. 5 | |
| POLR2A | 30.2 | 30.3 | 30.5 | 30.2 | 29.7 |
| N L63 gene | Ct-free | Ct-free | Ct-free | Ct-free | Ct-free |
In conclusion, the PO L R2A gene screened by the invention is proved to be capable of being applied to PCR diagnosis quality control of the respiratory RNA virus for the first time, and through designing a primer and a probe sequence aiming at the gene, an amplified fragment spans 2 exons, only the mRNA of the gene to be detected is specifically identified, the mRNA does not cross react with human genome DNA, namely the mRNA is only amplified, and the DNA is not amplified.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The embodiments and features of the embodiments of the present application may be combined with each other arbitrarily without conflict.
Sequence listing
<110> Saijian biological medicine (Shanghai) GmbH
<120> reference gene for respiratory RNA virus PCR detection and detection product thereof
<130>DD08153A2
<160>108
<170>SIPOSequenceListing 1.0
<210>1
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
aggataagag agccacgaac ca 22
<210>2
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
tcttgctgcc agtctggact gt 22
<210>3
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
gtgtgcacag gagccaagag tgaa 24
<210>4
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
<210>5
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
ttttcttgct gccagtctgg ac 22
<210>6
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
tgtgcacagg agccaagagt gaaga 25
<210>7
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
gataagagag ccacgaacc 19
<210>8
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
<210>9
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
tgcacaggag ccaagagtga agaac 25
<210>10
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
atccacagtg aatcttggtt g 21
<210>11
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>11
ttatcctcat gattaccgca gca 23
<210>12
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
acctaaagac cattgcactt cgt 23
<210>13
<211>16
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>13
ggcaatgcgg ctgcaa 16
<210>14
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>14
gggtacccac gcgaatcac 19
<210>15
<211>27
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>15
ctcatctttg ggctgttttc ttccgcc 27
<210>16
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>16
<210>17
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>17
accaactgtg ggtcatcct 19
<210>18
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>18
gagaccatgc aggctacca 19
<210>19
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>19
tgaaatcatt gctatgtcc 19
<210>20
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>20
ggctgttttc ttccgccgtt g 21
<210>21
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>21
ctccagggca tgttcaag 18
<210>22
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>22
<210>23
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>23
gttgttgatc tcgttctgtc 20
<210>24
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>24
<210>25
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>25
<210>26
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>26
tagtaggcat ggtaagggtc a 21
<210>27
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>27
tgaagctagg atccgacaga ac 22
<210>28
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>28
CATGTCGGGTTATTCGAGTGAC 19
<210>29
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>29
<210>30
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>30
TACTTCCTCCAAATCGAGGTG 19
<210>31
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>31
AAGCTCCTATTCTGATTGCTAC 22
<210>32
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>32
GAAGAACTGCTCGCAGTACCA 23
<210>33
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>33
CATCAATTATGACTACCCTAACTCCT 22
<210>34
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>34
CCAAGTTCAACCAAGCCAT 22
<210>35
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>35
TCACATTCTTGGCAGACACA 22
<210>36
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>36
ACCTGCCACCCAGATGACC 21
<210>37
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>37
GTGCTCCGTATTCGCATCATGA 22
<210>38
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>38
TCTGTCAGCATGTTGGACTCGAT 21
<210>39
<211>26
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>39
<210>40
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>40
<210>41
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>41
gttgaagtag agaccttcc 19
<210>42
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>42
ctacatcgtg cgagctaggc atggt 25
<210>43
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>43
ctgccaacaa cggagcattg c 21
<210>44
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>44
ggtgggtggc tcgcagtag 19
<210>45
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>45
tcagctacat cgtgcgagct aggc 24
<210>46
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>46
caggatgagt atgattggct g 21
<210>47
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>47
tccaagagaa gttgctcatc at 22
<210>48
<211>29
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>48
agacacctac tgaggctcct gcagactgc 29
<210>49
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>49
<210>50
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>50
cagttccaag agaagttgct cat 23
<210>51
<211>26
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>51
tgaggctcct gcagactgca gagcct 26
<210>52
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>52
<210>53
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>53
ggtatatgac aatggttct 19
<210>54
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>54
tggatcaagc tagatcagca ttctc 25
<210>55
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>55
cagagcgtcg ggatatcggg tg 22
<210>56
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>56
atatgacaat ggttctccac caa 23
<210>57
<211>26
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>57
atcaagctag atcagcattc tctaac 26
<210>58
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>58
cgaagttgcg gattgcagc 19
<210>59
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>59
cacatggtca gagactataa tc 22
<210>60
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>60
tcctacaaga ttatcaattt tgcc 24
<210>61
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>61
agttgcggat tgcagccgag aa 22
<210>62
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>62
tgtcaccatg ttcttcaggt a 21
<210>63
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>63
agtctctgac catgtggaat tcc 23
<210>64
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>64
agatttggac ctgcgagcg 19
<210>65
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>65
<210>66
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>66
ttctgacctg aaggctctgc gcg 23
<210>67
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>67
ccctgtgggt tttacactta a 21
<210>68
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>68
acgattgtgc atcagctga 19
<210>69
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>69
ccgtctgcgg tatgtggaaa ggttatgg 28
<210>70
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>70
ggggaacttc tcctgctaga at 22
<210>71
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>71
cagacatttt gctctcaagc tg 22
<210>72
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>72
<210>73
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>73
gcaacgcgcg attcagtt 18
<210>74
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>74
<210>75
<211>26
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>75
ctcttcacat aatcgccccg agctcg 26
<210>76
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>76
ggcactgagg acccacgtt 19
<210>77
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>77
ttgcgacata cccataaaag ca 22
<210>78
<211>27
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>78
ccccaaattg ctgagcttgc tcctaca 27
<210>79
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>79
gggtgtacct cttaatgcca attc 24
<210>80
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>80
<210>81
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>81
acccctgcgc aaaatgctgg g 21
<210>82
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>82
gaagtaccat ctggggctga g 21
<210>83
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>83
ccgaagagct acccgacg 18
<210>84
<211>26
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>84
ctctttcatt ttgccgtcac caccac 26
<210>85
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>85
tgtaggcact gattcaggtt ttg 23
<210>86
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>86
cggcgtggtc tgtatttaat tta 23
<210>87
<211>27
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>87
ctgcatacaa ccgctaccgt attggaa 27
<210>88
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>88
gaccratcct gtcacctctg ac 22
<210>89
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>89
agggcattyt ggacaaakcg tcta 24
<210>90
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>90
tgcagtcctc gctcactggg cacg 24
<210>91
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>91
aacagatgta agcagctccg ttatc 25
<210>92
<211>26
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>92
cgatttttat tggatgctgt acattt 26
<210>93
<211>26
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>93
tgccatagca tgacacaatg gctcct 26
<210>94
<211>26
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>94
aaaaacttag ggttaaagac aatcca 26
<210>95
<211>26
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>95
gccagatgtr tgtcyttcct gctggt 26
<210>96
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>96
caaacgatgg ctgaaaaagg ga 22
<210>97
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>97
catataagca tgctatatta aaagagtctc 30
<210>98
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>98
cctatttctg cagcatattt gtaatcag 28
<210>99
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>99
tgyaatgatg agggtgtcac tgcggttg 28
<210>100
<211>16
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>100
agccygcgtg gtgccc 16
<210>101
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>101
gaaacacgga cacccaaagt agt 23
<210>102
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>102
ctccggcccc tgaatgyggc taa 23
<210>103
<211>16
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>103
ggcggcctac tcatgg 16
<210>104
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>104
agactcttca caccttgttc atgt 24
<210>105
<211>16
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>105
aaaaccatga gacgct 16
<210>106
<211>26
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>106
aggaccttaa attcagacaa cgttct 26
<210>107
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>107
gattacgttt gcgattacca agact 25
<210>108
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>108
tgttgtttgg gttgctaag 19
Claims (8)
1. An internal reference gene for PCR detection of respiratory RNA viruses, which is PO L R2A gene.
2. The reference gene for PCR detection of respiratory RNA virus of claim 1, wherein the respiratory RNA virus comprises any one of severe acute respiratory syndrome virus, middle east respiratory syndrome virus, novel coronavirus, influenza virus, respiratory syncytial virus, parainfluenza virus, human metapneumovirus, human rhinovirus and human enterovirus.
3. The reference gene for PCR detection of respiratory RNA virus of claim 1, wherein the PCR detection comprises any one of the gene amplification technologies of reverse transcription-real-time fluorescence quantitative PCR detection, reverse transcription-general PCR detection, reverse transcription-loop-mediated isothermal amplification detection, reverse transcription-dependent nucleic acid sequence amplification detection, reverse transcription-rolling loop amplification detection, reverse transcription-single primer isothermal amplification detection, reverse transcription-dependent helicase DNA isothermal amplification, reverse transcription-recombinase polymerase amplification, reverse transcription-strand displacement amplification, and the like.
4. A primer for detecting the reference gene of claim 1, wherein the primer sequence of the PO L R2A gene is shown as SEQ ID NO.37 and SEQ ID NO. 38.
5. A probe for detecting the reference gene of claim 1, wherein the probe sequence of the PO L R2A gene is shown as SEQ ID NO. 39.
6. A PCR detection product for respiratory RNA viruses, which is characterized by comprising a primer and a probe for detecting the PO L R2A gene of the reference gene of claim 1, wherein the primer sequence of the PO L R2A gene is shown as SEQ ID NO.37 and SEQ ID NO.38, and the probe sequence is shown as SEQ ID NO. 39.
7. The PCR detection product for respiratory RNA virus of claim 6, which comprises a detection kit and a detection test paper.
8. The method for screening an internal reference gene for PCR detection of respiratory RNA virus of claim 1, comprising the steps of:
A. carrying out homology analysis on related genes, and screening genes with the pseudogene factor of 1 or less as candidate genes;
B. analyzing the expression level of each candidate gene in healthy nasopharyngeal tissue and skin tissue to infer the expression level thereof in a pharyngeal swab sample, and selecting a candidate gene expressed in at least one tissue;
C. and (3) carrying out expression stability analysis on the candidate gene, and screening to obtain a gene which can be stably detected in the population, namely the reference gene.
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| CN202010275432.9A Pending CN111394516A (en) | 2020-03-19 | 2020-04-09 | Internal reference gene for respiratory tract RNA virus PCR detection and detection product thereof |
| CN202010276167.6A Pending CN111363848A (en) | 2020-03-19 | 2020-04-09 | Internal reference gene for respiratory tract RNA virus PCR detection and detection product thereof |
| CN202010275441.8A Pending CN111286560A (en) | 2020-03-19 | 2020-04-09 | Internal reference gene for respiratory tract RNA virus PCR detection and detection product thereof |
| CN202010275417.4A Pending CN111455103A (en) | 2020-03-19 | 2020-04-09 | Internal reference gene for respiratory tract RNA virus PCR detection and detection product thereof |
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| CN202010275441.8A Pending CN111286560A (en) | 2020-03-19 | 2020-04-09 | Internal reference gene for respiratory tract RNA virus PCR detection and detection product thereof |
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| CN111394516A (en) | 2020-07-10 |
| CN111455103A (en) | 2020-07-28 |
| CN111286560A (en) | 2020-06-16 |
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