CN110724764A - Fluorescent quantitative PCR detection method for human coronavirus and respiratory syncytial virus and application thereof - Google Patents
Fluorescent quantitative PCR detection method for human coronavirus and respiratory syncytial virus and application thereof Download PDFInfo
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Abstract
The invention provides a primer and a probe or a combination thereof for fluorescent quantitative PCR (polymerase chain reaction) for detecting diseases caused by human coronavirus and respiratory syncytial virus. In specific embodiments, the primers and probes have the sequences set forth in SEQ ID NOs: 1-3, and/or as shown in SEQ ID NOs: 4-6, and/or as shown in SEQ ID NOs: 7-9. The invention also provides application of the primer and the probe or the combination thereof in preparing a reagent for detecting the human coronavirus OC43, the human coronavirus HKU1 and/or the respiratory syncytial virus RSVB in a sample.
Description
Technical Field
The invention relates to the field of human respiratory virus detection, in particular to a fluorescent quantitative PCR method and application thereof in the field of disease detection caused by human coronavirus and respiratory syncytial virus.
Background
At present, infection remains a great threat to human health, particularly acute respiratory infectious diseases, and due to the characteristics of multiple types, quick transmission, wide prevalence and the like, the incidence and death rate are high, and the serious threat to human health, social stability and economic development is caused. Global Disease, injury and risk factor Burden (GBD) studies found that Lower Respiratory tract infections in 2016 resulted in death in children under 65 million and 70 million adults, while about 238 of all ages worldwide died from Lower Respiratory tract infections, making Lower Respiratory tract infections the sixth leading cause of death in all ages, and also the leading cause of death in children under 5 years (see GBD 2016 Lower Respiratory tract infections. animals of the national institutions of the world, regional, and national sorbent, sport, and clinical of the systemic infections in 195 centers, 1990 + fungal 2016: a systematic analysis for the Global construct of the systemic Study 2016.Lance input disks 2018, 18 + 1210). Respiratory tract infections have been the leading cause of death and disability worldwide. Viruses are the leading cause of respiratory infections, with more than 50% of respiratory infections being caused by respiratory viruses. In addition, new viruses or variant strains are continuously appeared due to frequent respiratory virus variation, and new outbreaks/emergent epidemics are often caused. Respiratory viruses have become the focus of global infectious disease control and research, and efforts to reduce death caused by lower respiratory infection are priorities of countries in the world, so that improvement of the capability of detecting respiratory infection pathogens and coping with epidemic situations is urgently needed.
Coronaviruses (Coronavirus) belong to the family of coronaviridae, the genus coronaviruses, and infect mammals and birds, causing various diseases of the respiratory system, digestive system and central nerve [ see: hamre D, et al.A new viral associated from the human respiratory track, Proc Soc Exp Biol Med, 1966, 121 (1): 190-3.]. Coronaviruses can be divided into four different genera based on genomic and serological differences: α, β, γ and δ, only α and β genus coronaviruses currently infect humans. Up to now 6 human coronaviruses (HCoV) from two genera (α and β) have been identified, the α genus coronaviruses including NL63 and 229E, and the β genus coronaviruses including OC43, HKU1, acute respiratory syndrome coronavirus (SARS-CoV), and middle east respiratory syndrome coronavirus (MERS-CoV). HCoV is an important pathogen causing acute respiratory infections, which can be transmitted by direct contact with respiratory secretions or via aerosol droplets, and presents clinical manifestations ranging from mild colds to severe lower respiratory infections. It has been found that about 3.36% of acute lower respiratory infections in children in our country are caused by coronaviruses [ see Qian Y, et al, detection and clinical analysis of aerobic respiratory tract infection with human coronaviruses in childrenin Beiijing area 2007-2015, Zhonghua Er Ke Za Zhi 2015 Sep; 53(9): 707-11]. Respiratory Syncytial Virus (RSV), a single-stranded RNA virus of the genus pneumovirus of the family paramyxoviridae, is classified into two types A, B, and is the leading cause of respiratory infections in infants under 5 years of age, and further, RSV infects people of various age groups and causes severe respiratory diseases, the condition of which is particularly severe among the elderly. In hospitalization and outpatient/emergency cases under 2 years old in China, the RSV detection rate is high in leaderboard, and reaches 33.3% [ see Yu J, et al, Complex of the prediction of respiratory diseases in patients with access resources in North China, 2012-2015, BMC Infect Dis.2018 Feb 8; 18(1): 72].
The severe clinical disease burden is caused by human coronavirus and respiratory syncytial virus, and the development of a sensitive, rapid, specific, stable and convenient diagnostic method is urgently needed, so that the clinical application can provide basis for clear pathogeny and early diagnosis, promote early reasonable treatment, reduce the drug resistance burden increased by the use of empirical antibiotics and control the disease condition in time.
The detection method for respiratory viruses mainly comprises virus separation, antigen detection, antibody detection (such as enzyme-linked immunosorbent assay and immunofluorescence analysis), a traditional Polymerase Chain Reaction (PCR) method, a fluorescent quantitative PCR (Real-time PCR) method and the like. PCR is a molecular biology technique, and specific DNA fragments are amplified in vitro without depending on organisms such as Escherichia coli or yeast. It is a simple and reliable method for copying DNA fragments, and is suitable for many fields of modern biology and related science. The PCR detection method is rapid and sensitive, shows strong advantages for viruses which can not be cultured, are difficult to be cultured or have long culture time, and is particularly suitable for detecting the acute-phase infection of the viruses when the antigens are difficult to detect due to antigen diversity or low antigen content of clinical samples. The fluorescent quantitative PCR technique is a method for detecting the total amount of products after PCR cycles in a DNA amplification reaction by using oligonucleotides modified with fluorescent substances (such as FAM, HEX, ROX, CY5, etc.) at the 5 'end and quenching substances (such as TAMRA, etc.) at the 3' end. When PCR reaction is carried out, a fluorescent probe is added into the PCR reaction solution, the fluorescent probe is hybridized with the template in the annealing process of the PCR reaction, and the fluorescent substance at the 5 'end is restricted by the quenching substance at the 3' end and can not emit fluorescence. During the extension process of the further PCR reaction, the 5 '→ 3' Exonuclease (exouclase) activity of the TaqDNA polymerase decomposes the fluorescent probe hybridized with the template, causing the free fluorescent substance to fluoresce. The purpose of detecting the amplification amount of the PCR product is achieved by detecting the fluorescence intensity in the reaction system. The fluorescent quantitative PCR technology has unique advantages in nucleic acid detection, such as easy operation, high accuracy, high sensitivity, strong specificity, fast detection time, high flux, difficult pollution and the like, so the fluorescent quantitative PCR technology is widely applied to detection in clinic and scientific research laboratories.
The conventional PCR method designs corresponding primers for each pathogen, and one reaction detects one pathogen, which is limited when it is required to detect multiple pathogens. In view of this, multiplex pcr (multiplex pcr) technology is developed, and multiple target genes can be amplified simultaneously in one system, so as to realize simultaneous detection of multiple viral pathogens and meet clinical requirements. There is still a need to design primers with better performance to detect various respiratory viruses rapidly, specifically, sensitively and accurately.
Disclosure of Invention
In the invention, specific primers and probes aiming at OC43, HKU1 and RSVB genes are successfully designed, and the respiratory viruses OC43, HKU1 and RSVB can be successfully detected respectively by extracting nucleic acid from a respiratory tract sample of a patient infected by the acute respiratory virus and then detecting the nucleic acid. Further combining primers and probes for three pathogens of respiratory tract infection, OC43, HKU1 and RSVB can be detected simultaneously in samples of acute respiratory tract infection. The primer and the probe in the detection system have better sensitivity and specificity, and the detection system has better repeatability.
In a first aspect, the present invention provides primers and probes for detecting human coronavirus OC43 in a sample by a fluorescent quantitative PCR method, wherein the primers are used for specifically amplifying an OC43N protein gene sequence, and the probes specifically bind to an OC43N protein gene internal sequence.
In a second aspect, the present invention provides primers and probes for detecting human coronavirus HKU1 in a sample by a fluorescent quantitative PCR method, the primers being used for specifically amplifying the gene sequence of HKU1N protein, and the probes specifically binding to the internal sequence of the gene of HKU1N protein.
In a third aspect, the invention provides a primer and a probe for detecting the respiratory syncytial virus RSVB in a sample by a fluorescent quantitative PCR method, wherein the primer is used for specifically amplifying the RSVB N protein gene sequence, and the probe is specifically combined with the internal sequence of the RSVB N protein gene.
In a fourth aspect, the present invention provides a primer and probe combination for simultaneous detection of human coronavirus OC43, human coronavirus HKU1 and/or respiratory syncytial virus RSVB in a sample by a fluorescent quantitative PCR method, said primer and probe combination consisting of or consisting of the primers and probes of the first and second aspects, or of the second and third aspects, or of the first, second and third aspects.
In a fifth aspect, the present invention provides a fluorescent quantitative PCR kit comprising the primers and probes of any one of the first to third aspects, or the primer and probe combination of the fourth aspect.
In a sixth aspect, the invention provides the use of the primer and probe of any one of the first to third aspects, or the primer and probe combination of the fourth aspect, for the preparation of a reagent for detecting human coronavirus OC43, human coronavirus HKU1 and/or respiratory syncytial virus RSVB in a sample.
Drawings
FIG. 1 shows the detection of OC43 by fluorescent quantitative PCR.
FIG. 2 shows the detection of HKU1 by fluorescent quantitative PCR method.
FIG. 3 shows the detection of RSVB by the fluorescent quantitative PCR method.
FIG. 4 shows simultaneous detection of OC43, HKU1 and RSVB by fluorescent quantitative PCR.
FIG. 5 shows the detection limit analysis of the fluorescent quantitative PCR method: (A) OC 43; (B) HKU 1; (C) RSVB.
Detailed Description
The invention provides a group of primers and probes for detecting human coronavirus OC43 in a sample by a fluorescent quantitative PCR method, wherein the primers are used for specifically amplifying an OC43N protein gene sequence, and the probes are specifically combined with an OC43N protein gene internal sequence; preferably, the nucleotide sequences of the primers and probes are as shown in SEQ ID NOs: 1-3.
The invention also provides a group of primers and probes for detecting the human coronavirus HKU1 in a sample by a fluorescent quantitative PCR method, wherein the primers are used for specifically amplifying the gene sequence of the HKU1N protein, and the probes are specifically combined with the internal sequence of the gene of the HKU1N protein; preferably, the nucleotide sequences of the primers and probes are as shown in SEQ ID NOs: 4-6.
The invention also provides a group of primers and probes for detecting the respiratory syncytial virus RSVB in the sample by a fluorescent quantitative PCR method, wherein the primers are used for specifically amplifying the RSVB N protein gene sequence, and the probes are specifically combined with the internal sequence of the RSVBN protein gene; preferably, the nucleotide sequences of the primers and probes are as shown in SEQ ID NOs: 7-9.
The invention also provides a primer and a probe group for simultaneously detecting the human coronavirus OC43, the human coronavirus HKU1 and/or the respiratory syncytial virus RSVB in a sample by a fluorescent quantitative PCR method, wherein the primer and the probe group consist of any two groups of the specific primers and the probes or consist of the three groups of the specific primers and the probes; preferably, the sequences of the primer and probe sets are as set forth in SEQ ID NOs: 1-6, or SEQ ID NOs: 4-9, or SEQ ID NOs: 1-3 and 7-9, or SEQ id nos: 1-9.
The invention also provides a fluorescent quantitative PCR kit, which comprises the primers and the probes of any group, or the primers and the probes of any two groups, or the three groups of primers and the probes.
In a specific embodiment, the kit further comprises an enzyme mixture, a PCR buffer, and nuclease-free water.
The invention also provides application of the primers and the probes or the combination thereof in preparing a reagent for detecting the human coronavirus OC43, the human coronavirus HKU1 and/or the respiratory syncytial virus RSVB in a sample.
In a specific embodiment, the sample is a clinical sample of a patient with a respiratory infection; preferably, the sample comprises sputum, nasopharyngeal swab, alveolar lavage.
The invention will be further illustrated with reference to preferred embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Specific experimental procedures are not noted in the examples below, and are generally performed according to conventional conditions and methods, such as those described in the molecular cloning laboratory Manual (Sambrook, et al. New York: Cold Spring harbor laboratory Press, 1989) or those provided by the reagent manufacturer.
Example 1 fluorescent quantitative PCR method for detecting human coronavirus OC43
1.1 extraction of nucleic acids
Clinical samples of patients with acute respiratory infection, such as sputum, nasopharyngeal swab, alveolar lavage fluid, etc., are taken, and nucleic acid is extracted according to a conventional method. The obtained nucleic acid was dissolved in 50. mu.l of an eluent, and the amount and purity of the extracted nucleic acid were determined by a conventional method. Subpackaging the extracted nucleic acid into small parts, and freezing and storing in a refrigerator at-80 ℃.
1.2 design and Synthesis of specific primers and probes
A Primer 3 software is utilized to respectively design a fluorescent quantitative PCR forward Primer and a fluorescent quantitative PCR reverse Primer aiming at an OC43N protein gene sequence, and a probe is designed in the gene. The design method comprises the following steps: aiming at a targeted target gene, a sliding window method is used for designing a TaqMan probe, the window width is set to be 120 nucleotides, the step length is 1 nucleotide, and all possible probe sequences are exhausted. The probes were screened using the following conditions: 1) the length of the probe sequence is 20-35 nucleotides; 2) the Tm value of the probe sequence is 70 +/-2 ℃; 3) the probe does not form a hairpin structure with the virus gene; 4) the GC content of the probe sequence is 40-70%. After the TaqMan probe is determined, a primer matched with the TaqMan probe is designed by using software, and the primer needs to meet the following conditions: 1) the length is 18-35 nucleotides, and the difference between the lengths of the upstream primer and the downstream primer is not more than 4 nucleotides; 2) the Tm value of the primer is 60 +/-2 ℃, and the difference between the Tm values of the upstream primer and the downstream primer is not more than 2 ℃; 3) the primer does not form a circular hairpin structure; 4) the primers do not form dimers by themselves; 5) no dimer is formed between the upstream primer and the downstream primer; 6) the 3' end of the primer is not G or C; 7) the amplicon size of the upstream and downstream primers is 80-180 nucleotides. And designing a plurality of pairs of primers and carrying out a matching experiment on the designed probes, and selecting the primer pair with the lowest Ct value.
Synthesizing a primer and a probe according to a conventional method, and adding a FAM fluorescent group at the 5' end of the probe. The forward primer, reverse primer and probe were named OC43N For, OC43N Rev, OC43N FAM, respectively. The primers and probes were made up to 10. mu.M with sterile water without DNase and RNase, aliquoted into small portions and frozen in a freezer at-20 ℃.
1.3 preparation of fluorescent quantitative PCR System
AgPath-ID of ABI Life TechnologiesTMThe One-Step RT-PCR Kit was used as an example to prepare a fluorescent quantitative PCR system.
AgPath-IDTMOne-Step RT-PCR Kit reagent, primer and probeTaking out the mixture in a refrigerator at the temperature of-20 ℃, placing the mixture on ice after dissolving, and preparing an RT-PCR master mix system in a PCR tube:
the nucleic acid was removed from the freezer at-80 ℃ and adjusted for concentration, and 2. mu.l (1-50ng) of the nucleic acid was added to the PCR tube.
1.4 fluorescent quantitative PCR reaction procedure
The prepared fluorescent quantitative PCR reaction system is put into a fluorescent quantitative PCR instrument, and the following procedures are executed:
1.5 determination of results
And (3) judging standard: negative control no amplification curve; sample CT< 35 and typical "S" type amplification curves.
The results show that OC43 positive samples can be specifically detected in acute respiratory infection samples, as shown in figure 1.
Example 2 fluorescent quantitative PCR method for detecting human coronavirus HKU1
2.1 extraction of nucleic acids
Clinical samples of patients with acute respiratory infection, such as sputum, nasopharyngeal swab, alveolar lavage fluid, etc., are taken. The nucleic acid was extracted according to a conventional method, and the obtained nucleic acid was dissolved in 50. mu.l of the eluate. The extracted nucleic acid is quantified and purity checked according to conventional methods. Subpackaging the extracted nucleic acid into small parts, and freezing and storing in a refrigerator at-80 ℃.
2.2 design and Synthesis of specific primers and probes
A Primer 3 software is utilized to respectively design a fluorescent quantitative PCR forward Primer and a fluorescent quantitative PCR reverse Primer aiming at the gene sequence of the HKU1N protein, and a probe is designed in the gene. The procedure is described in 1.2. Primers and probes were synthesized according to the conventional method, and ROX fluorophore was added to the 5' end of the probe. The forward primer, reverse primer and probe were named HKU1N For, HKU1N Rev, HKU1N FAM, respectively. The primers and probes were made up to 10. mu.M with sterile water without DNase and RNase, aliquoted into small portions and frozen in a freezer at-20 ℃.
2.3 preparation of fluorescent quantitative PCR System
AgPath-ID of ABI Life TechnologiesTMThe One-Step RT-PCR Kit was used as an example to prepare a fluorescent quantitative PCR system.
AgPath-IDTMTaking out the One-Step RT-PCR Kit reagent, the primer and the probe from a refrigerator at the temperature of-20 ℃, dissolving the reagent, the primer and the probe, placing the dissolved reagent on ice, and preparing an RT-PCR master mix system in a PCR tube:
| composition of | 1×25(μl) |
| Nuclease-free water | 8 |
| 2 × RT-PCR buffer | 12.5 |
| HKU1 For | 0.5 |
| HKU1 Rev | 0.5 |
| HKU1 ROX | 0.5 |
| RT-PCR enzyme mixtures | 1 |
| Total up to | 23 |
The nucleic acid was removed from the freezer at-80 ℃ and adjusted for concentration, and 2. mu.l (1-50ng) of the nucleic acid was added to the PCR tube.
2.4 fluorescent quantitative PCR reaction procedure
The prepared fluorescent quantitative PCR reaction system is put into a fluorescent quantitative PCR instrument, and the following procedures are executed:
2.5 determination of results
And (3) judging standard: negative control no amplification curve; sample CT< 35 and typical "S" type amplification curves.
The results showed that HKU1 positive samples could be specifically detected in the acute respiratory infection samples, as shown in FIG. 2.
Example 3 detection of respiratory syncytial virus RSVB by fluorescent quantitative PCR method
3.1 extraction of nucleic acids
Clinical samples of patients with acute respiratory infection, such as sputum, nasopharyngeal swab, alveolar lavage fluid, etc., are taken, nucleic acid is extracted according to a conventional method, and the obtained nucleic acid is dissolved in 50 μ l of eluent. The extracted nucleic acid is quantified and purity checked according to conventional methods. Subpackaging the extracted nucleic acid into small parts, and freezing and storing in a refrigerator at-80 ℃.
3.2 design and Synthesis of specific primers and probes
And respectively designing a fluorescent quantitative PCR forward Primer and a fluorescent quantitative PCR reverse Primer aiming at the RSVB N protein gene sequence by using Primer 3 software, and designing a probe in the gene. The procedure is described in 1.2. Primers and probes were synthesized according to the conventional method, and CY5 fluorophore was added to the 5' end of the probe. The forward, reverse and probe primers are designated RSVBN For, RSVBN Rev, RSVBN CY5, respectively. The primers and probes were made up to 10. mu.M with sterile water without DNase and RNase, aliquoted into small portions and frozen in a freezer at-20 ℃.
3.3 preparation of fluorescent quantitative PCR System
AgPath-ID of ABI Life TechnologiesTMThe One-Step RT-PCR Kit was used as an example to prepare a fluorescent quantitative PCR system.
AgPath-IDTMTaking out the One-Step RT-PCR Kit reagent, the primer and the probe from a refrigerator at the temperature of-20 ℃, dissolving the reagent, the primer and the probe, placing the dissolved reagent on ice, and preparing an RT-PCR master mix system in a PCR tube:
| composition of | 1×25(μl) |
| Nuclease-free water | 8 |
| 2 × RT-PCR buffer | 12.5 |
| RSVB For | 0.5 |
| RSVB Rev | 0.5 |
| RSVB CY5 | 0.5 |
| 25 × RT-PCR enzyme mixture | 1 |
| Total up to | 23 |
The nucleic acid was removed from the freezer at-80 ℃ and adjusted for concentration, and 2. mu.l (1-50ng) of the nucleic acid was added to the PCR tube.
3.4 fluorescent quantitative PCR reaction procedure
Putting the prepared fluorescent quantitative PCR reaction system into a fluorescent quantitative PCR instrument, and executing the following procedures:
3.5 determination of results
And (3) judging standard: negative control no amplification curve; sample CT< 35 and typical "S" type amplification curves.
The results show that RSVB positive samples can be specifically detected in acute respiratory tract infection samples, and the results are shown in figure 3.
Example 4 fluorescent quantitative PCR method for simultaneous detection of OC43, HKU1 and RSVB
4.1 extraction of nucleic acids
Clinical samples of patients with acute respiratory infection, such as sputum, nasopharyngeal swab, alveolar lavage fluid, etc., are taken, and nucleic acid is extracted according to a conventional method. The resulting nucleic acid was dissolved in 50. mu.l of the eluent. The extracted nucleic acid is quantified and purity checked according to conventional methods. Subpackaging the extracted nucleic acid into small parts, and freezing and storing in a refrigerator at-80 ℃.
4.2 OC43, HKU1 and RSVB specific primers and probes
The primer and probe sequences specific for OC43, HKU1 and RSVB were the corresponding primer and probe sequences of examples 1, 2 and 3, respectively, and were dissolved in DNase-and RNase-free sterile water after synthesis, all primers and probes were mixed, and the final concentration of each primer or probe in the primer-probe mixture was adjusted to 10. mu.M. Subpackaging the mixed solution into small parts, and freezing and storing in a refrigerator at the temperature of-20 ℃.
4.3 preparation of fluorescent quantitative PCR System
AgPath-ID of ABI Life TechnologiesTMThe One-Step RT-PCR Kit was used as an example to prepare a fluorescent quantitative PCR system.
AgPath-IDTMTaking out the One-Step RT-PCR Kit reagent, the primer and the probe from a refrigerator at the temperature of-20 ℃, dissolving the reagent, the primer and the probe, placing the dissolved reagent on ice, and preparing an RT-PCR master mix system in a PCR tube:
| composition of | 1×25(μl) |
| Nuclease-free water | 8.5 |
| 2 × RT-PCR buffer | 12.5 |
| Primer-probe mixture | 1 |
| 25 × RT-PCR enzyme mixture | 1 |
| Total up to | 23 |
The nucleic acid was removed from the freezer at-80 ℃ and adjusted for concentration, and 2. mu.l (1-50ng) of the nucleic acid was added to the PCR tube.
4.4 fluorescent quantitative PCR reaction procedure
Putting the prepared fluorescent quantitative PCR reaction system into a fluorescent quantitative PCR instrument, and executing the following procedures:
4.5 determination of results
And (3) judging standard: negative control no amplification curve; sample CT< 35 and typical "S" type amplification curves.
The results show that in the same detection system, the acute respiratory tract infection sample can specifically detect OC43, HKU1 and RSVB positive samples at the same time, as shown in FIG. 4.
Example 5 fluorescent quantitative PCR method for the detection of specificity and sensitivity assays for OC43, HKU1 and RSVB
The invention takes a nested PCR method (hereinafter referred to as nested PCR) of a nucleic acid detection scheme of fever respiratory syndrome virus in a national science and technology major infectious disease monitoring platform network laboratory as a gold standard, and compares the OC43, HKU1 and RSVB fluorescent quantitative PCR methods with the nested PCR method. And taking a sample which is positive through gold standard nested PCR detection and has correct sequencing as a positive reference, and randomly selecting and analyzing 500 detection negative samples. The results suggest that the specificity of the OC43 fluorescent quantitative PCR method was 100% and the sensitivity was 100% using nested PCR as gold standard (table 1); the specificity of the HKU1 fluorescent quantitative PCR method was 100%, and the sensitivity was 100% (Table 2); the specificity of the RSVB fluorescent quantitative PCR method was 99.6%, and the sensitivity was 98% (Table 3).
TABLE 1 specificity and sensitivity analysis of the OC43 fluorescent quantitative PCR method
Note: sensitivity is a/(a + b); specificity d/(c + d)
TABLE 2 specificity and sensitivity analysis of HKU1 fluorescent quantitative PCR method
Note: sensitivity is a/(a + b); specificity d/(c + d)
TABLE 3 specificity and sensitivity analysis of the RSVB fluorescent quantitative PCR method
Note: sensitivity is a/(a + b); specificity d/(c + d)
Example 6 repeatability (precision) analysis of the fluorescent quantitative PCR detection methods of OC43, HKU1 and RSVB
Randomly sampling acute respiratory infection samples positive to OC43, HKU1 and RSVB virus, extracting nucleic acid, quantifying, and taking 106Copying/reacting on the same test day and repeating 5 holes on the same instrument to perform fluorescent quantitative PCR analysis, and detecting the cycle threshold (CT value); randomly extracting OC43, HKU1 and RSVB virus positive acute respiratory infection specimen, extracting nucleic acid, quantifying 106The copy/reaction was analyzed by fluorescent quantitative PCR on different test days, and CT was detected for 5 replicates. The mean and standard deviation were calculated separately, and the intra-and inter-batch Coefficient of Variation (CV) was calculated from the standard deviation/mean. The results show that the OC43, HKU1 and RSV within batch CVs were 0.18%, 0.09% and 0.25%, respectively, the inter-batch CV values were 0.15%, 0.17% and 0.24%, respectively, and both the intra-and inter-batch CV values were less than 1%, indicating good reproducibility (table 4).
TABLE 4 repeatability (precision) analysis of the fluorescent quantitative PCR detection methods of OC43, HKU1 and RSVB
Example 7 analysis of detection limits of the fluorescent quantitative PCR detection methods for OC43, HKU1 and RSVB
The OC43, HKU1 and RSV target fragments were PCR amplified, cloned into plasmids containing T7 promoter, further transcribed into RNA in vitro, residual DNA was digested with DNase, and corresponding copy number was calculated according to RNA concentration and fragment size. And (3) performing gradient dilution on the RNA, respectively performing fluorescent quantitative PCR analysis by using the RNA as a template, and making a standard curve according to the gradient copy number and the corresponding Ct value. And performing fluorescent quantitative PCR analysis on OC43, HKU1 and RSVB positive samples respectively, and sleeving the Ct value of the samples into a standard curve to obtain the copy number of the viral RNA. Then 10 is put8The copy/reaction OC43, HKU1 and RSVB positive samples are subjected to 10-fold gradient dilution and used as templates for carrying out fluorescence quantitative PCR analysis, and the lowest detected copy numbers of the reaction system on OC43, HKU1 and RSVB are respectively 3, 2 and 5. The results are shown in the graph SA-C.
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Claims (9)
1. Primers and probes for detecting human coronavirus OC43 in a sample by a fluorescent quantitative PCR method, wherein the primers are used for specifically amplifying an OC43N protein gene sequence, and the probes are specifically combined with an OC43N protein gene internal sequence; preferably, the nucleotide sequences of the primers and probes are as shown in SEQ ID NOs: 1-3.
2. Primers and probes for detecting human coronavirus HKU1 in a sample by a fluorescent quantitative PCR method, wherein the primers are used for specifically amplifying HKU1N protein gene sequences, and the probes are specifically combined with HKU1N protein gene internal sequences; preferably, the nucleotide sequences of the primers and probes are as shown in SEQ ID NOs: 4-6.
3. The primers and the probes are used for detecting the respiratory syncytial virus RSVB in a sample by a fluorescent quantitative PCR method, the primers are used for specifically amplifying the RSVB N protein gene sequence, and the probes are specifically combined with the internal sequence of the RSVB N protein gene; preferably, the nucleotide sequences of the primers and probes are as shown in SEQ ID NOs: 7-9.
4. Primer and probe combination for the simultaneous detection of human coronavirus OC43, human coronavirus HKU1 and/or respiratory syncytial virus RSVB in a sample by a fluorescent quantitative PCR method, said primer and probe combination consisting of the primers and probes of claims 1 and 2, or consisting of the primers and probes of claims 2 and 3, or consisting of the primers and probes of claims 1, 2 and 3; preferably, the sequence of the primer and probe combination is as shown in SEQ ID NOs: 1-6, or SEQ ID NOs: 4-9, or SEQ ID NOs: 1-3 and 7-9, or SEQ ID NOs: 1-9.
5. A fluorescent quantitative PCR kit comprising the primer and probe of any one of claims 1-3, or the primer and probe combination of claim 4.
6. The kit according to claim 5, further comprising an enzyme mixture, a PCR buffer and nuclease-free water.
7. Use of the primers and probes of any one of claims 1-3 or the primer and probe combination of claim 4 for the preparation of a reagent for the detection of human coronavirus OC43, human coronavirus HKU1 and/or respiratory syncytial virus RSVB in a sample.
8. The use of claim 7, wherein the sample is a clinical sample from a patient with a respiratory infection.
9. The use of claim 8, wherein the sample comprises sputum, nasopharyngeal swab, alveolar lavage.
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