CN112410469A - Fluorescent RT-PCR reagent and method for detecting influenza A virus, influenza B virus and coronavirus SARS-CoV-2 - Google Patents
Fluorescent RT-PCR reagent and method for detecting influenza A virus, influenza B virus and coronavirus SARS-CoV-2 Download PDFInfo
- Publication number
- CN112410469A CN112410469A CN202011320537.8A CN202011320537A CN112410469A CN 112410469 A CN112410469 A CN 112410469A CN 202011320537 A CN202011320537 A CN 202011320537A CN 112410469 A CN112410469 A CN 112410469A
- Authority
- CN
- China
- Prior art keywords
- virus
- influenza
- cov
- reagent
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a freeze-dried multiplex fluorescence RT-PCR reagent and a method for detecting and distinguishing influenza A virus, influenza B virus and coronavirus SARS-CoV-2. The primer and probe for fluorescent RT-PCR detection reagent of influenza A virus, influenza B virus and coronavirus SARS-CoV-2 provided by the invention are designed and screened based on the latest gene sequence information of influenza A virus, influenza B virus and coronavirus SARS-CoV-2 in public gene sequence database, and have good inclusion, specificity and reaction performance. The reagent adopts human RNase P nucleic acid fragment as a detection internal standard. In addition, the invention prepares the influenza A virus, influenza B virus and coronavirus SARS-CoV-2 fluorescent RT-PCR detection reagent into a freeze-dried reagent which can be stored at normal temperature by a freeze-drying technology, avoids the risk of failure of the conventional liquid fluorescent PCR detection reagent due to storage temperature change or repeated freeze thawing, and reduces the transportation and management costs.
Description
Technical Field
The invention relates to a pathogenic microorganism detection technology, in particular to nucleic acid detection of influenza A virus, influenza B virus and coronavirus SARS-CoV-2.
Background
Respiratory infections are the most common group of diseases in humans and are among the leading causes of morbidity and mortality in the population. Studies have shown that respiratory viruses are the most common causative agents of respiratory diseases. Respiratory viruses include influenza, coronavirus, rhinovirus, respiratory syncytial virus, parainfluenza virus, human metapneumovirus, adenovirus, and the like. The clinical manifestations caused by respiratory virus infection are similar, mainly including rhinitis, pharyngitis, laryngitis, tonsillitis, tracheitis, bronchitis, pneumonia, cough, dyspnea, fever, hypodynamia, etc., but the treatment methods, curative effects and disease courses are different from each other for infections caused by different pathogens.
Influenza viruses are the causative agents of influenza. Influenza viruses belong to the family orthomyxoviridae and are classified into types a, b, c and d, which were first discovered in 2016. The genome of influenza viruses is composed of seven or eight negative-strand RNA segments, each of which encodes one or two proteins. The influenza A virus can be divided into 16H subtypes (H1-H16) and 9N subtypes (N1-N9) according to different antigenicity of outer membrane Hemagglutinin (HA) and Neuraminidase (NA) proteins of virus particles, and because nucleotide sequences for coding the HA and the NA are easy to mutate, epitope of the HA and the NA is changed, the original specific immunity of people is disabled, and the influenza A virus often causes large-scale epidemics. Influenza b viruses are mainly classified into Victoria and Yamagata systems, and have small antigenic variation and can cause local outbreaks of influenza. The C type rarely causes epidemic, and the D type has not been reported to be epidemic. In the field of medical health, diagnosis and monitoring of influenza a and b viruses is of utmost importance.
Coronaviruses are another major group of respiratory viruses. Coronaviruses belong to the order of nested viruses and the family of coronaviruses, and are classified into four genera, alpha, beta, gamma and delta, and genomes are composed of linear single-stranded positive-stranded RNA. It has now been found that 7 coronaviruses, namely HCoV-229E and HCoV-NL63 of the genus α, and HCoV-OC43, HCoV-HKU1, SARS-CoV, MERS-CoV and SARS-CoV-2 of the genus β, infect humans. Wherein SARS-CoV-2 is the pathogen causing the epidemic situation of the current novel coronavirus pneumonia (COVID-19), and has the closest relationship with Bat SARS coronavirus (Bat SARS-like coronavirus) and SARS-CoV. COVID-19 can cause similar symptoms of pneumonia, cough, fever, hypodynamia and the like as influenza, so that the method has important significance for distinguishing SARS-CoV-2 from influenza virus in clinical diagnosis and epidemic prevention and control.
The fluorescence RT-PCR technology uses a specific respiratory virus gene sequence as a detection target to carry out in-vitro qualitative detection on respiratory virus nucleic acid from a human body sample. The fluorescent RT-PCR has been increasingly applied to the clinical auxiliary diagnosis of respiratory virus infection related diseases due to the characteristics of accuracy, sensitivity, rapidness and the like, and is the main diagnosis method of COVID-19 at present. However, the conventional fluorescent RT-PCR detection reagent is a liquid reagent, the detection activity can be maintained only by storing at the temperature of below-20 ℃, and the detection performance of the reagent is reduced by the temperature rise and repeated freeze thawing in the processes of storage, transportation and use. Particularly, the transportation is blocked in the epidemic situation period, the cold chain transportation condition is difficult to guarantee, the risk of reagent failure and the cold chain management cost are greatly increased in the long-distance transportation process, and the method cannot adapt to the international long-distance transportation process and the application of the basic level detection mechanism with poor cold chain condition.
In addition, due to global outbreaks, different regions enter the winter influenza season separately, and influenza presents greater challenges for epidemic control because it resembles the early symptoms of COVID-19. The market urgently needs a fluorescent RT-PCR detection reagent which can accurately detect and distinguish influenza virus and coronavirus SARS-CoV-2.
Disclosure of Invention
The purpose of the invention is: provides a multiplex fluorescence RT-PCR detection reagent and a method which can accurately detect and distinguish influenza A virus, influenza B virus and coronavirus SARS-CoV-2 and does not need a cold chain.
The technical problem to be solved by the invention is realized by the following technical scheme:
1. design of primers and probes
The primer probe used by the SARS-CoV-2 fluorescence RT-PCR detection reagent for influenza A virus, influenza B virus and coronavirus is obtained by designing and screening based on the latest nucleic acid information of the influenza A virus, the influenza B virus and the SARS-CoV-2 in a public gene sequence database. Preferably, a primer probe specific to human RNase P is added to the reagent as an internal standard for detection. The sequences of the primer probes are as follows:
the sequence of the specific forward primer for detecting the influenza A virus is 5 '-CATGGARTGGCTAAAGACAAG ACC-3' (namely SEQ ID No.1), the sequence of the reverse primer is 5'-CATTTTGGACAAAGCGTCTACG-3' (namely SEQ ID No.2), and the sequence of the probe is 5'-CAGTCCTCGCTCACTGGGCACG-3' (namely SEQ ID No. 3). The sequences of the primers and the probes are highly conserved in the nucleic acid sequences of the known influenza A viruses, and have exclusivity to other species.
The sequences of two specific forward primers for detecting influenza B virus are 5'-TTCGAGCGTCTTAATGAA GGACA-3' (SEQ ID No.4) and 5'-CGAGCGTCTCAATGAAGGACA-3' (SEQ ID No.5) (the two forward primers are designed aiming at the single nucleotide polymorphism at the position and can be used after being mixed according to a certain proportion), the sequence of a reverse primer is 5'-TGGTGATAATCGGTGCTCTTGAC-3' (SEQ ID No.6), and the sequence of a probe is 5'-CAAAGCCAATTCGAGCAGCTGAAACTG-3' (SEQ ID No. 7). The sequences of the primers and the probes are highly conserved in the known influenza B virus nucleic acid sequences and have exclusivity to other species.
The sequence of the specific forward primer for detecting coronavirus SARS-CoV-2 is 5'-TGCAGTCATAACAAGA GAAGTGGGT-3' (i.e. SEQ ID No.8), the sequence of the reverse primer is 5'-CAAAAAGTCACCATTA GTTGTGCG-3' (i.e. SEQ ID No.9), and the sequence of the probe is 5'-TTGTCGTGCCTGGTTTGCCTG G-3' (i.e. SEQ ID No. 10). The nucleic acid sequences of the primer and the probe are highly conserved in the known SARS-CoV-2 nucleic acid sequence, and have exclusivity to the nucleic acid sequences of the recent Bat SARS-like coronavirus and SARS-CoV.
The sequence of the specific forward primer of the internal standard human RNase P is 5'-CTGCGAGCGGGTTCTGAC-3' (i.e. SEQ ID No.11), the sequence of the reverse primer is 5'-AACGATATGATTGATAGCAACAACTGA-3' (i.e. SEQ ID No.12), and the sequence of the probe is 5'-AAGGCTCTGCGCGGACTTGTGGA-3' (i.e. SEQ ID No. 13). The sequence of the primer and the probe is highly conserved in the nucleic acid sequence of the known human RNase P and has exclusivity to other species.
The 5 'end of the probe for detecting influenza A virus, influenza B virus, coronavirus SARS-CoV-2 and human RNase P is modified by a fluorescence reporter group, and the 3' end is modified by a fluorescence quenching group. The fluorescent reporter groups of the probes of the three viruses and the human RNase P are respectively selected from different FAM, JOE, HEX, VIC, TET, ROX and CY5, and the fluorescent quenching groups are respectively selected from any one of BHQ1, BHQ2, BHQ3, TAMRA and Eclipse. The fluorescence reporter group of the influenza A virus probe is preferably FAM, and the fluorescence quencher group is preferably BHQ 1; the fluorescence reporter group of the influenza B virus probe is preferably JOE, and the fluorescence quencher group is preferably BHQ 2; the fluorescence reporter group of the coronavirus SARS-CoV-2 probe is preferably ROX, and the fluorescence quencher group is preferably BHQ 2; the fluorescence reporter group of the human RNase P probe is preferably CY5, and the fluorescence quencher group is preferably BHQ 2.
2. Establishment of reaction System
The reaction volume of the fluorescent RT-PCR detection reagent for the influenza A virus, the influenza B virus and the coronavirus SARS-CoV-2 is 25 mul, the reagent comprises all reaction components (shown in table 1) except sample nucleic acid required by the fluorescent RT-PCR detection for the influenza A virus, the influenza B virus and the coronavirus SARS-CoV-2, and the detection can be carried out on a computer by adding water and the sample nucleic acid to be detected to the reaction volume of 25 mul.
TABLE 1 influenza A, B and coronavirus SARS-CoV-2 fluorescent RT-PCR assay reagent composition
3. Lyophilization of reagents
The invention prepares the influenza A virus, influenza B virus and coronavirus SARS-CoV-2 fluorescent RT-PCR detection reagent into a freeze-dried reagent which is stable at normal temperature by a vacuum freeze-drying technology, thereby avoiding the risk of invalidation of the conventional liquid fluorescent PCR detection reagent due to the change of storage temperature or repeated freeze thawing.
The freeze-dried fluorescent RT-PCR detection reagent for influenza A virus, influenza B virus and coronavirus SARS-CoV-2 is prepared by freeze-drying the components in the table 1, the reagent can be freeze-dried in a container, and preferably, the reagent can be freeze-dried in a PCR tube or a multi-connected tube which can be directly arranged on a machine according to 1 part of the reagent per tube. The freeze dried fluorescent RT-PCR reagent for influenza A virus, influenza B virus and coronavirus SARS-CoV-2 is white particle and may be stored at normal temperature in light shielding and damp proof condition for 12 months. The volume of dry matter contained in a single reagent is about 2 mu l, and when the reagent is used, the water with the total volume of 23 mu l and the nucleic acid of the sample to be detected are directly added to prepare a 25 mu l reaction system. Preferably, the volumes of the added water and the sample nucleic acid are 18. mu.l and 5. mu.l, respectively. As another preference, in the final elution step of the sample nucleic acid extraction, a suitable concentration of sample nucleic acid can be obtained by appropriately increasing the amount of the eluent (e.g., 100. mu.l), and 23. mu.l of the sample nucleic acid can be directly added to a single lyophilized reagent. Adding water and nucleic acid sample into the reagent, slowly shaking to dissolve the reagent, centrifuging for a short time, and detecting on machine.
4. Reaction procedure and result determination
The reaction program of the fluorescent RT-PCR detection reagent for influenza A virus, influenza B virus and coronavirus SARS-CoV-2 is set according to the table 2, and the fluorescent signal is set according to the fluorescent reporter group of the probe of each detection target.
TABLE 2 reaction procedure for influenza A, B and coronavirus SARS-CoV-2 fluorescent RT-PCR detection reagents
After reaction, the Ct of the negative control (no nuclease water) is more than or equal to 40 or no Ct, the Ct of the influenza A virus, the influenza B virus and SARS-CoV-2 in the positive control is less than or equal to 35, and the Ct of the RNase P internal standard (when the RNase P internal control is contained in the reagent) in the sample is less than 40, so the detection is effective. Otherwise, the result is invalid, and the error factor is removed and then the result is rechecked. If the detection is effective, the detection results of the influenza A virus, the influenza B virus and the SARS-CoV-2 are judged according to the following conditions respectively:
a) the Ct of the detection sample is less than or equal to 35, and the detection sample can report that the pathogen is positive;
b) the Ct of the detected sample is more than 35 and less than 40, the detected sample is reported to be suspected to be positive by the pathogen, and the detection needs to be repeated or determined by an independent method.
c) If the Ct of the detected sample is more than or equal to 40 or no Ct, the pathogen is reported to be negative.
The invention has the beneficial effects that:
the primer and probe sequence used by the freeze-dried fluorescent RT-PCR detection reagent for the influenza A virus, the influenza B virus and the coronavirus SARS-CoV-2 are designed and screened based on the latest nucleic acid sequence information of the influenza A virus, the influenza B virus and the coronavirus SARS-CoV-2, have good inclusion, specificity and reaction performance, can ensure the detection accuracy and distinguish the influenza A virus, the influenza B virus and the coronavirus SARS-CoV-2. In addition, the invention uses vacuum freeze drying technology to prepare the influenza A virus, influenza B virus and coronavirus SARS-CoV-2 fluorescent RT-PCR detection reagent into a freeze-dried reagent without cold chain, thereby avoiding the risk of failure of the conventional liquid fluorescent PCR detection reagent due to storage temperature change or repeated freeze thawing, and being particularly suitable for being used in regions with poor long-distance transportation and cold chain conditions.
Drawings
FIG. 1 shows the target sequence for detection of influenza A virus and the positions of specific primers and probes according to the present invention.
FIG. 2 shows the target sequence for detecting influenza B virus and the positions of specific primers and probes according to the present invention.
FIG. 3 shows the detection target sequence of coronavirus SARS-CoV-2 and the positions of specific primers and probes.
FIG. 4 shows the detection target sequence of human RNase P and the positions of specific primers and probes according to the present invention.
FIG. 5 shows the morphology of fluorescent RT-PCR detection reagents for influenza A, B and coronavirus SARS-CoV-2 lyophilized in PCR octant tubing.
FIG. 6 shows the detection results and the repeatability of the lyophilized fluorescent RT-PCR detection reagent for influenza A virus, influenza B virus and coronavirus SARS-CoV-2 to the positive reference nucleic acid of influenza A virus, influenza B virus, SARS-CoV-2 and RNase P.
FIG. 7 shows the linear relationship between the amplification efficiency of the lyophilized fluorescent RT-PCR detection reagent for influenza A virus, influenza B virus and coronavirus SARS-CoV-2 and different concentrations.
FIG. 8 shows the linear relationship between the amplification efficiency of the lyophilized fluorescent RT-PCR detection reagent for influenza A virus, influenza B virus and coronavirus SARS-CoV-2 and different concentrations.
FIG. 9 shows the linear relationship between the amplification efficiency and different concentrations of the lyophilized fluorescent RT-PCR detection reagent for influenza A virus, influenza B virus and coronavirus SARS-CoV-2 nucleic acid positive reference substance.
FIG. 10 shows the linear relationship between the amplification efficiency of the lyophilized fluorescent RT-PCR detection reagent for human RNase P nucleic acid positive reference and different concentrations for influenza A virus, influenza B virus and coronavirus SARS-CoV-2 according to the present invention.
Detailed Description
The following examples are merely illustrative of the present invention, and the scope of the present invention shall be subject to the scope of the claims.
Example 1: this example provides a design scheme of fluorescent RT-PCR primers and probes for detecting influenza A, B, SARS-CoV-2 and human RNase P nucleic acids.
The invention selects the nucleic acid sequences shown in figures 1, 2, 3 and 4 as the detection target sequences of influenza A virus, influenza B virus, coronavirus SARS-CoV-2 and human RNase P respectively by analyzing the genome information of influenza A virus, influenza B virus and coronavirus SARS-CoV-2 and the nucleic acid sequence information of human RNase P in a public gene sequence database. The following primers for amplifying a target sequence and probe sequences for detecting a target sequence were designed using Primer Express V3.0 software:
the sequence of the specific forward primer of influenza a virus is SEQ ID No.1, Tm 59.0 ℃, the sequence of the reverse primer is SEQ ID No.2, Tm 58.4 ℃, the sequence of the probe is SEQ ID No.3, Tm 68.2 ℃. The 5 'end of the probe is modified by a fluorescence reporter group FAM, and the 3' end of the probe is modified by a fluorescence quenching group BHQ 1. The amplicon has a length of 122bp, and the sequence has no similarity with nucleic acid sequences of other species determined by BLAST examination, namely has good specificity and is beneficial to preventing false detection. The primer and probe sequences are highly conserved among known influenza A virus strains, which is beneficial to preventing missed detection.
The sequences of two specific forward primers of influenza B virus are SEQ ID No.4(Tm is 59.8 ℃) and SEQ ID No.5(Tm is 60.0 ℃), the sequence of a reverse primer is SEQ ID No.6, Tm is 60.0 ℃, the sequence of a probe is SEQ ID No.7, and Tm is 68.3 ℃. The 5 'end of the probe is modified by a fluorescence reporter group JOE, and the 3' end of the probe is modified by a fluorescence quenching group BHQ 2. The length of the amplicon is 99bp or 97bp, and the sequence is determined by B LAST inspection to have no similarity with nucleic acid sequences of other species, namely, the sequence has good specificity and is beneficial to preventing false detection. The primer and probe sequences are highly conserved among known influenza B virus strains, which is favorable for preventing omission.
The sequence of the specific forward primer of SARS-CoV-2 is SEQ ID No.8, Tm is 60.0 deg.C, the sequence of the reverse primer is SEQ ID No.9, Tm is 59.6 deg.C, the sequence of the probe is SEQ ID No.10, and Tm is 68.0 deg.C. The 5 'end of the probe is modified by a fluorescence reporter group ROX, and the 3' end of the probe is modified by a fluorescence quenching group BHQ 2. The length of the amplicon is 82bp, and the sequences have enough difference with the sequences of the Bat SARS-like coronavirus and SARS-CoV with the highest homology determined by BLAST examination, namely, the sequences have good specificity and are beneficial to preventing false detection. The primer and probe sequences are highly conserved in the known SARS-CoV-2 strain, which is favorable for preventing omission.
The sequence of the specific forward primer of human RNase P is SEQ ID No.11, Tm 59.1 ℃, the sequence of the reverse primer is SEQ ID No.12, Tm 59.0 ℃, the sequence of the probe is SEQ ID No.13, Tm 69.8 ℃. The 5 'end of the probe is modified by a fluorescence reporter group Cy5, and the 3' end of the probe is modified by a fluorescence quenching group BHQ 2. The amplicon has a length of 93bp, and the sequence has no similarity with nucleic acid sequences of other species determined by BLAST examination, namely has good specificity and is beneficial to preventing false detection. The primer and probe sequences are highly conserved in the known human RNase P sequence, which is beneficial to preventing missed detection.
The GC contents of the forward primer, the reverse primer and the probe of the influenza A virus, the influenza B virus, the coronavirus SARS-CoV-2 and the RNase P internal standard are all in a reasonable range; the Tm difference between the primers is small, which is beneficial to the simultaneous annealing of the primers; the Tm of the probe is higher than that of the primer by more than 8 ℃, and the difference is large enough, so that the probe can complete annealing earlier than the primer. The self structures of the primer and the probe are well maintained, and stable secondary structures such as self dimer, pair dimer and hairpin pin which reduce the reaction efficiency cannot be formed. Shorter amplicons also contribute to increased PCR reaction efficiency.
The forward primer, the reverse primer and the probe are synthesized by Huada gene, and are prepared into 100uM of preservation solution and 10uM of working solution by TE buffer solution to be preserved at the temperature of minus 20 ℃.
Example 2: this example demonstrates that the fluorescent RT-PCR detection reagent for influenza A virus, influenza B virus and coronavirus SARS-CoV-2 of the present invention is prepared into lyophilized reagent by vacuum freeze-drying technique.
Influenza A virus, influenza B virus and coronavirus SARS-CoV-2 fluorescent RT-PCR liquid detection reagents are prepared according to the components and the content in the table 1, and are packaged into 200 mul PCR eight-connected tubes according to 1 part per tube for vacuum freeze drying, and the obtained influenza A virus, influenza B virus and coronavirus SARS-CoV-2 freeze-drying type fluorescent RT-PCR detection reagents are white particles (figure 5). Add 23. mu.l nuclease-free water to one reagent, shake slowly for 10s, the reagent is completely dissolved, the volume measured by pipette is 25. mu.l, and the volume before re-dissolving is equivalent to 2. mu.l.
In order to prevent the reagent from being affected with damp and exposed, the lyophilized fluorescent RT-PCR detection reagent of influenza A virus, influenza B virus and coronavirus SARS-CoV-2 is firstly treated with silica gel desiccant in vacuum, then packaged in an aluminum foil bag and stored at normal temperature.
Example 3: this example demonstrates the detection effect of the lyophilized fluorescent RT-PCR detection reagent for influenza A virus, influenza B virus and coronavirus SARS-CoV-2 on the positive reference nucleic acid of influenza A virus, influenza B virus, SARS-CoV-2 and human RNase P.
10-fold gradient dilution is carried out on the mixed liquid of positive reference products of influenza A virus, influenza B virus, SARS-CoV-2 and human RNase P RNA, 5 mul and 18 mul of nuclease-free water are mixed for each concentration, and then the mixture is added into 1 part of freeze-dried fluorescent RT-PCR detection reagent of influenza A virus, influenza B virus and coronavirus SARS-CoV-2 prepared according to the embodiment 2; mu.l nuclease-free water was used as a negative control. The reaction mixture of 25. mu.l volume was obtained, and was subjected to slow shaking dissolution, centrifugation and detection on a fluorescence PCR machine, and the reaction program was set as in Table 2. Each treatment was repeated 3 times.
The detection result is shown in figure 6, the freeze-drying fluorescence RT-PCR detection reagent for the influenza A virus, the influenza B virus and the coronavirus SARS-CoV-2 has positive detection results for the influenza A virus, the influenza B virus, the SARS-CoV-2 and the human RNase P nucleic acid positive reference substance with no less than 10 copies, the difference Coefficient (CV) of Ct among the copies is less than 5 percent, and the negative reference substance has no amplification signal. The common logarithm of the template concentration and the Ct thereof have a good linear relationship (the correlation coefficients for influenza A virus, influenza B virus, SARS-CoV-2 and human RNase P are-0.999, -0.998 and-0.996 respectively), and the amplification efficiencies of the nucleic acids of the influenza A virus, the influenza B virus, SARS-CoV-2 and human RNase P are 98.27%, 101.76%, 93.65% and 93.44% respectively (FIGS. 7, 8, 9 and 10).
It can be seen that the freeze-dried fluorescent RT-PCR detection reagent for influenza A virus, influenza B virus and coronavirus SARS-CoV-2 of the invention has good detection performance for influenza A virus, influenza B virus, SARS-CoV-2 and human RNase P nucleic acid, and has a plurality of advantages simultaneously: (1) can be preserved at normal temperature without the requirement of the traditional liquid reagent on the preservation temperature (-20 ℃); (2) before use, the reagent does not need to be thawed, so that the influence of repeated freezing and thawing on the activity of the reagent is completely avoided; (3) the reagent is subpackaged according to single parts, and liquid preparation and subpackaging steps are not needed during use, so that the reagent loss and the risk of reagent pollution are reduced.
Sequence listing
<110> Shenzhen Seikanol Biotech Ltd
<120> fluorescent RT-PCR reagent and method for detecting influenza A virus, influenza B virus and coronavirus SARS-CoV-2
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213> artificial sequence
<400> 1
catggartgg ctaaagacaa gacc 24
<210> 2
<211> 22
<212> DNA
<213> artificial sequence
<400> 2
<210> 3
<211> 22
<212> DNA
<213> artificial sequence
<400> 3
<210> 4
<211> 23
<212> DNA
<213> artificial sequence
<400> 4
ttcgagcgtc ttaatgaagg aca 23
<210> 5
<211> 21
<212> DNA
<213> artificial sequence
<400> 5
cgagcgtctc aatgaaggac a 21
<210> 6
<211> 23
<212> DNA
<213> artificial sequence
<400> 6
tggtgataat cggtgctctt gac 23
<210> 7
<211> 27
<212> DNA
<213> artificial sequence
<400> 7
caaagccaat tcgagcagct gaaactg 27
<210> 8
<211> 25
<212> DNA
<213> artificial sequence
<400> 8
tgcagtcata acaagagaag tgggt 25
<210> 9
<211> 24
<212> DNA
<213> artificial sequence
<400> 9
caaaaagtca ccattagttg tgcg 24
<210> 10
<211> 22
<212> DNA
<213> artificial sequence
<400> 10
ttgtcgtgcc tggtttgcct gg 22
<210> 11
<211> 18
<212> DNA
<213> artificial sequence
<400> 11
<210> 12
<211> 27
<212> DNA
<213> artificial sequence
<400> 12
aacgatatga ttgatagcaa caactga 27
<210> 13
<211> 23
<212> DNA
<213> artificial sequence
<400> 13
aaggctctgc gcggacttgt gga 23
Claims (4)
1. A multiple fluorescence RT-PCR reagent for detecting influenza A virus, influenza B virus and coronavirus SARS-CoV-2 is characterized in that the reagent is a freeze-dried reagent, comprises all necessary components of the fluorescence RT-PCR except sample nucleic acid, and is prepared by a vacuum freeze-drying method by using Tris-HCl buffer solution, potassium chloride, magnesium chloride, dNTPs, Taq DNA polymerase, reverse transcriptase, a freeze-drying excipient, and specific primers and probes for detecting influenza A virus, influenza B virus and coronavirus SARS-CoV-2.
2. The multiplex fluorescence RT-PCR reagent for detecting influenza A virus, influenza B virus and coronavirus SARS-CoV-2 as claimed in claim 1, wherein the specific primers and probe sequences for detecting influenza A virus, influenza B virus and coronavirus SARS-CoV-2 are as follows:
the forward primer sequence for detecting the influenza A virus is SEQ ID No.1, the reverse primer sequence is SEQ ID No.2, and the probe sequence is SEQ ID No. 3;
detecting influenza B virus, wherein the forward primer sequence is SEQ ID No.4 and SEQ ID No.5, the reverse primer sequence is SEQ ID No.6, and the probe sequence is SEQ ID No. 7;
the forward primer sequence for detecting the coronavirus SARS-CoV-2 is SEQ ID No.8, the reverse primer sequence is SEQ ID No.9, and the probe sequence is SEQ ID No. 10;
the 5 'end of the probe for detecting influenza A virus, influenza B virus and coronavirus SARS-CoV-2 is modified by a fluorescence reporter group, the 3' end of the probe is modified by a fluorescence quenching group, the fluorescence reporter groups of the probes of the three viruses are respectively selected from different FAM, JOE, HEX, VIC, TET, ROX and CY5, and the fluorescence quenching group is any one of BHQ1, BHQ2, BHQ3, TAMRA and Eclipse.
3. The multiplex fluorescence RT-PCR reagent for detecting influenza A virus, influenza B virus and coronavirus SARS-CoV-2 as claimed in claim 1 or 2, characterized in that it further comprises specific forward primer, reverse primer and probe of human RNase P as internal detection standard, the sequence of the forward primer of human RNase P is SEQ ID No.11, the sequence of the reverse primer is SEQ ID No.12, and the sequence of the probe is SEQ ID No. 13;
the 5 'end and the 3' end of the RNase P probe are respectively modified by a fluorescence reporter group and a fluorescence quencher group, wherein the fluorescence reporter group is selected from FAM, JOE, HEX, VIC, TET, ROX and CY5 and is different from the fluorescence reporter group of the probe for influenza A virus, influenza B virus and coronavirus SARS-CoV-2, and the quencher group is any one of BHQ1, BHQ2, BHQ3, TAMRA and Eclipse.
4. The multiplex fluorescent RT-PCR reagent for detecting influenza A virus, influenza B virus and coronavirus SARS-CoV-2 as claimed in claim 1, 2 or 3, wherein 1 part of each tube is lyophilized in a PCR reaction tube which can be directly used in the machine or a multi-connected tube composed of the PCR reaction tube, when in use, the reagent is not required to be thawed, water and the nucleic acid of the sample to be detected are added, and the reagent can be used in the machine for detection after short shaking and centrifugation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011320537.8A CN112410469A (en) | 2020-11-23 | 2020-11-23 | Fluorescent RT-PCR reagent and method for detecting influenza A virus, influenza B virus and coronavirus SARS-CoV-2 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011320537.8A CN112410469A (en) | 2020-11-23 | 2020-11-23 | Fluorescent RT-PCR reagent and method for detecting influenza A virus, influenza B virus and coronavirus SARS-CoV-2 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112410469A true CN112410469A (en) | 2021-02-26 |
Family
ID=74778086
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011320537.8A Pending CN112410469A (en) | 2020-11-23 | 2020-11-23 | Fluorescent RT-PCR reagent and method for detecting influenza A virus, influenza B virus and coronavirus SARS-CoV-2 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112410469A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113621735A (en) * | 2021-08-11 | 2021-11-09 | 江苏金迪克生物技术股份有限公司 | Method for detecting influenza virus titer by fluorescent quantitative PCR |
| WO2022210122A1 (en) * | 2021-03-29 | 2022-10-06 | 東洋紡株式会社 | Set of oligonucleotides for detecting plurality of types of viruses through multiplex pcr |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3339451A1 (en) * | 2015-08-21 | 2018-06-27 | Mmonitor Inc. | Primers for detecting influenza by using lamp, and use thereof |
| CN110578017A (en) * | 2019-07-30 | 2019-12-17 | 深圳市百迈生命科学有限公司 | Kit for synchronously detecting twenty-three respiratory pathogens and detection method thereof |
| CN111088408A (en) * | 2020-03-20 | 2020-05-01 | 广州凯普医药科技有限公司 | Detection kit for new coronavirus, influenza A and influenza B and respiratory syncytial virus |
| CN111411172A (en) * | 2020-02-28 | 2020-07-14 | 江苏硕世生物科技股份有限公司 | Probe and primer composition for simultaneously detecting novel human coronavirus, influenza A virus and influenza B virus |
| CN111719016A (en) * | 2020-05-25 | 2020-09-29 | 深圳市疾病预防控制中心 | Composition for detecting new coronavirus 2019-nCoV and influenza A and B viruses and application |
| CN111961762A (en) * | 2020-09-03 | 2020-11-20 | 深圳市赛格诺生物科技有限公司 | Fluorescent RT-PCR primer, probe, reagent and method for detecting coronavirus SARS-CoV-2 |
-
2020
- 2020-11-23 CN CN202011320537.8A patent/CN112410469A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3339451A1 (en) * | 2015-08-21 | 2018-06-27 | Mmonitor Inc. | Primers for detecting influenza by using lamp, and use thereof |
| CN110578017A (en) * | 2019-07-30 | 2019-12-17 | 深圳市百迈生命科学有限公司 | Kit for synchronously detecting twenty-three respiratory pathogens and detection method thereof |
| CN111411172A (en) * | 2020-02-28 | 2020-07-14 | 江苏硕世生物科技股份有限公司 | Probe and primer composition for simultaneously detecting novel human coronavirus, influenza A virus and influenza B virus |
| CN111088408A (en) * | 2020-03-20 | 2020-05-01 | 广州凯普医药科技有限公司 | Detection kit for new coronavirus, influenza A and influenza B and respiratory syncytial virus |
| CN111719016A (en) * | 2020-05-25 | 2020-09-29 | 深圳市疾病预防控制中心 | Composition for detecting new coronavirus 2019-nCoV and influenza A and B viruses and application |
| CN111961762A (en) * | 2020-09-03 | 2020-11-20 | 深圳市赛格诺生物科技有限公司 | Fluorescent RT-PCR primer, probe, reagent and method for detecting coronavirus SARS-CoV-2 |
Non-Patent Citations (4)
| Title |
|---|
| NATHAMON NGAOSUWANKUL: "Influenza A viral loads in respiratory samples collected from patients infected with pandemic H1N1, seasonal H1N1 and H3N2 viruses", 《VIROLOGY JOURNAL》 * |
| 佚名: "GenBank 登录号：KC891620.1", 《NCBI》 * |
| 佚名: "GenBank 登录号：MT446351", 《NCBI》 * |
| 许静洪: "《生物化学实验指导》", 31 August 2010, 中国科学技术出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022210122A1 (en) * | 2021-03-29 | 2022-10-06 | 東洋紡株式会社 | Set of oligonucleotides for detecting plurality of types of viruses through multiplex pcr |
| CN113621735A (en) * | 2021-08-11 | 2021-11-09 | 江苏金迪克生物技术股份有限公司 | Method for detecting influenza virus titer by fluorescent quantitative PCR |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111254228B (en) | Kit for detecting novel coronavirus and influenza virus | |
| CN111020064B (en) | Novel coronavirus ORF1ab gene nucleic acid detection kit | |
| CN111004870B (en) | Novel coronavirus N gene nucleic acid detection kit | |
| CN111363858B (en) | 2019-nCoV S gene detection nucleic acid composition, kit and production method | |
| CN111808989A (en) | Coronavirus/influenza virus/rhinovirus nucleic acid combined detection kit and use method thereof | |
| CN111270017A (en) | Primer probe combination for detecting novel coronavirus based on digital PCR and application thereof | |
| US20230151444A1 (en) | Pcr based diagnostic kit, compositions and methods for amplification and detection of sars-cov-2 | |
| CN116171333A (en) | Compositions and methods for detecting severe acute respiratory syndrome coronavirus 2 (SARS-COV-2), influenza A and influenza B | |
| CN112410469A (en) | Fluorescent RT-PCR reagent and method for detecting influenza A virus, influenza B virus and coronavirus SARS-CoV-2 | |
| CN113005226A (en) | Oligonucleotide and kit for detecting SARS-CoV-2 | |
| Kondiah et al. | A Simple-Probe® real-time PCR assay for genotyping reassorted and non-reassorted isolates of Crimean-Congo hemorrhagic fever virus in southern Africa | |
| CN113652505A (en) | Method and kit for detecting novel coronavirus and VOC-202012/01 mutant strain thereof | |
| CN111961762A (en) | Fluorescent RT-PCR primer, probe, reagent and method for detecting coronavirus SARS-CoV-2 | |
| Zhang et al. | Rapid and accurate identification of SARS-CoV-2 variants containing E484 mutation | |
| CN113789411B (en) | Influenza A and B virus nucleic acid detection kit and use method thereof | |
| JP4950656B2 (en) | Nucleic acid detection | |
| CN112813202A (en) | Fluorescent RT-PCR (reverse transcription-polymerase chain reaction) detection reagent and method for coxsackievirus A16 and enterovirus 71 | |
| CN111363859B (en) | Nucleic acid composition for 2019-nCoV detection, kit and production method thereof | |
| JP2023536962A (en) | Compositions and methods for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-2), influenza A and influenza B | |
| Gorna et al. | Establishment and validation of two duplex one-step real-time RT-PCR assays for diagnosis of foot-and-mouth disease | |
| Ahmed et al. | Evaluating two approaches for using positive control in standardizing the avian influenza H5 reverse transcription recombinase polymerase amplification assay | |
| CN111304367A (en) | Freeze-dried fluorescent RT-PCR reagent and method for detecting 2019 novel coronavirus | |
| CN112662808A (en) | Novel coronavirus COVID-19 nucleic acid detection kit and detection method thereof | |
| CN111235319A (en) | Influenza A and B virus nucleic acid composite detection kit | |
| CN113073152A (en) | LAMP primer, probe and kit for detecting influenza B virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210226 |
|
| RJ01 | Rejection of invention patent application after publication |