CN114574630A - Kit for rapidly detecting multiple respiratory pathogens, probe and primer combination - Google Patents

Kit for rapidly detecting multiple respiratory pathogens, probe and primer combination Download PDF

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CN114574630A
CN114574630A CN202110733994.8A CN202110733994A CN114574630A CN 114574630 A CN114574630 A CN 114574630A CN 202110733994 A CN202110733994 A CN 202110733994A CN 114574630 A CN114574630 A CN 114574630A
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赵西浩
陈永红
张蓉
郭玉婉
李秀林
李伟伟
彭莉
苏波
刘中华
王国强
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Abstract

The invention discloses a kit for rapidly detecting multiple respiratory pathogens, which is mainly based on a melting curve technology and comprises specific primers and probes for 2019-nCoV, FluA, FluB, H3N2, RSVA, RSVB, HRV, HAdV3, HAdV7, HMPV and MP.

Description

Kit for rapidly detecting multiple respiratory pathogens, probe and primer combination
The technical field is as follows:
the invention belongs to the technical field of nucleic acid detection, and particularly relates to a kit for rapidly detecting multiple respiratory pathogens, a probe and primer combination and a preparation process thereof.
Background art:
the invention belongs to the technical field of nucleic acid detection, and particularly relates to a kit and a detection method for detecting novel coronavirus (2019-nCoV), influenza A virus, influenza B virus, respiratory syncytial virus A, respiratory syncytial virus B, rhinovirus, H3N2, adenovirus type 3, adenovirus type 7, metapneumovirus and mycoplasma pneumoniae.
Coronaviruses (Coronavirus, CoV) belong to the order nidoviridae, the family coronaviridae, and are transmitted mainly by direct contact with secretions or via aerosols, droplets, and there is also evidence for transmission via the fecal oral route. The clinical manifestations of the novel coronavirus pneumonia include fever, hypodynamia and other general symptoms accompanied by dry cough, dyspnea and the like, and the coronavirus pneumonia can rapidly develop into severe pneumonia, respiratory failure, acute respiratory distress syndrome, septic shock, multiple organ failure, severe acid-base metabolic disorder and the like, and even endanger life.
Influenza is an acute respiratory infectious disease caused by influenza virus, and the main mode of transmission is air droplet transmission. The protein is classified into four types, i.e., A, B, C and D (or A, B, C, D) according to the difference between the Nucleoprotein (NP) and the matrix protein (M). Influenza a viruses are classified into various subtypes according to protein structures and gene characteristics of Hemagglutinin (HA) and Neuraminidase (NA) on the surface of the viruses. The HA and NA found to date have 18 (H1-18) and 11 (N1-11) subtypes, respectively. Influenza a viruses are most susceptible to mutation and are likely to cause outbreaks; influenza b virus has less variation, influenza c virus is more stable, and sporadic cases are caused more often. Typical symptoms of influenza are marked by sudden fever, dizziness, headache and myalgia, and can be accompanied by symptoms such as sore throat, cough, nasal obstruction, watery nasal discharge, chest pain, eye pain, photophobia and the like.
Respiratory Syncytial Virus (RSV) belongs to the genus pneumovirus, and is classified into type A and type B according to the antigenicity of adhesion protein G, and the clinical symptoms caused by the type A and the type B are similar. RSV is the most main pathogen of infant lower respiratory tract infection, has the highest incidence rate in 2-6 months, has a latent period of 3-7 days, has serious symptoms, and can have high fever, rhinitis, pharyngitis and laryngitis, and then is manifested as bronchiolitis and pneumonia. The minority of sick children can be complicated by otitis media, pleuritis, myocarditis and the like. Premature birth, congenital heart disease, lung dysplasia and the like are major factors causing danger. RSV infection not only can induce asthma, but is also associated with exacerbations. After infection in adults and older children, upper respiratory tract infections are mainly manifested. RSV usually throttles in winter and spring and has a subtype distribution difference. The disease is transmitted via air droplets and intimate contact.
Human Rhinovirus (HRV) is a single-stranded positive-stranded RNA virus of the enterovirus genus, approximately 7.5kb in length, with only one open reading frame, A, B, C serotypes, and more than 150 genotypes. Mainly causes upper respiratory tract infection, causes lower respiratory tract infection by HRV of partial types, and particularly causes serious diseases for infants and elderly people who have immunosuppression. HRV enters the body through the nasal, oral, and ocular mucosae, mainly by contact and droplet transmission.
Human adenoviruses (HAdV) are DNA viruses and belong to the family adenoviridae. Adenovirus pneumonia accounts for about 4% -10% of community-acquired pneumonia, severe pneumonia is common in types 3 and 7, and respiratory discomfort is generally caused after infection. Depending on the type of adenovirus used, it may cause discomfort in the digestive tract, conjunctivitis, cystitis, exanthema, etc., of which respiratory diseases and infectious conjunctivitis are most common. Mainly via air droplets and intimate contact.
Human Metapneumovirus (HMPV) is a single negative strand enveloped RNA virus, approximately 13kb in length, containing 8 open reading frames of genes encoding 9 proteins in total. HMPV infection occurs well in infants and children, with major symptoms including upper respiratory infection, bronchiolitis, pneumonia, etc., and few infants without clinical symptoms. HMPV can develop all the year round, is prevalent in late winter, spring and early summer, and is transmitted by respiratory droplets, hand-mouth contact, hand-eye contact with the surface of pollutants.
Mycoplasma Pneumoniae (MP) is the causative agent of Mycoplasma Pneumoniae in humans. The pneumonia of the mycoplasma pneumoniae has slow onset of disease, and symptoms such as anorexia, nausea, vomit, pharyngalgia, headache, fever, muscular soreness and the like exist at the initial stage of disease. MP is mainly infected by droplets, the incubation period is 2-3 weeks, teenagers are high-incidence people, and the MP can occur all the year round, but mostly in autumn and winter.
The detection methods of respiratory pathogens at present are various, and mainly comprise virus culture, serological detection and molecular biological detection. Among them, virus culture and serological detection are easy to have false negative due to long time consumption, low sensitivity, high culture requirement. Common molecular biological methods include ordinary PCR, nested PCR, fluorescent quantitative PCR, isothermal amplification, and the like. For the detection of various pathogens in the respiratory tract, the detection is limited by a detection channel, and generally consists of a multi-tube reagent, so that the operation is complicated, the operation time is long, and the flux is also reduced.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
The invention content is as follows:
the invention aims to provide a kit for rapidly detecting multiple respiratory pathogens based on a melting curve, thereby overcoming the defects in the prior art.
In order to achieve the purpose, the invention provides a kit for rapidly detecting multiple respiratory pathogens based on a melting curve, which is characterized by comprising specific primers aiming at 2019-nCoV, FluA, FluB, H3N2, RSVA, RSVB, HRV, HAdV3, HAdV7, HMPV and MP, wherein the specific primers aiming at the N gene of 2019-nCoV are SEQ ID NO. 1-2, the specific primers aiming at ORF1ab gene of 2019-nCoV are SEQ ID NO. 3-4, the specific primers aiming at M gene of FluA are SEQ ID NO. 5-6, the specific primers aiming at N gene of FluB are SEQ ID NO. 7-8, the specific primers aiming at P gene of RSVA are SEQ ID NO. 9-10, the specific primers aiming at P gene of RSVB are SEQ ID NO. 11-12, the specific primers aiming at 5' UTR gene of HRV are SEQ ID NO. 13-3614, the specific primers aiming at N gene of HRV are SEQ ID NO. 3-2, the HEXON gene specific primers aiming at ADV3 are SEQ ID NO. 17-18, the HEXON gene specific primers aiming at ADV7 are SEQ ID NO. 19-20, the M gene specific primers aiming at HMPV are SEQ ID NO. 21-22, and the CARDS gene specific primers aiming at MP are SEQ ID NO. 23-24.
Preferably, in the technical scheme, the primer specific to RNaseP of human DNA internal reference is SEQ ID NO. 25-26.
A kit for rapidly detecting multiple respiratory pathogens based on a melting curve comprises specific probes for 2019-nCoV, FluA, FluB, H3N2, RSVA, RSVB, HRV, HAdV3, HAdV7, HMPV and MP, wherein the specific probe for the N gene of 2019-nCoV is SEQ ID NO.27, the specific probe for ORF1ab gene of 2019-nCoV is SEQ ID NO.28, the specific probe for the M gene of FluA is SEQ ID NO.29, the specific probe for the N gene of FluB is SEQ ID NO.30, the specific probe for the P gene of RSVA is SEQ ID NO.31, the specific probe for the P gene of RSVB is SEQ ID NO.32, the specific probe for the 5' UTR gene of HRV is SEQ ID NO.33, the specific probe for the HA gene of H3N2 is SEQ ID NO.34, the specific probe for the XON gene of ADV3 is SEQ ID NO.35, and the specific probe for the ADV gene 7 is SEQ ID NO.36, the M gene specific probe for HMPV is SEQ ID No.37, and the CARDS gene specific probe for MP is SEQ ID No. 38.
Preferably, in the above technical scheme, the specific probe for human ribonuclease P, RNaseP, which is a reference in human DNA, is SEQ ID NO. 39.
Preferably, in the above technical scheme, two ends of the specific probe respectively carry a fluorescent group and a quenching group, wherein the fluorescent group is selected from any one or more of FAM, HEX, VIC, TET, TAMRA, ROX, CY3.5 or CY 5; the quenching group is selected from any one or more of BHQ1, BHQ2, BHQ3, DABCYL and MGB.
A method for rapidly detecting multiple respiratory pathogens based on a melting curve comprises the step of adding a sample to be detected into an RT-PCR reaction system containing an RT-PCR primer probe to perform real-time fluorescent quantitative RT-PCR reaction, and is characterized in that the RT-PCR primer probe comprises an amplification primer pair and a detection probe respectively aiming at 2019-nCoV, FluA, FluB, H3N2, RSVA, RSVB, HRV, HAdV3, HAdV7, HMPV and MP; wherein: the specific primers for 2019-nCoV are SEQ ID NO. 1-2, the specific primers for 2019-nCoV are SEQ ID NO. 3-4, the specific primers for FluA are SEQ ID NO. 5-6, the specific primers for FluB are SEQ ID NO. 7-8, the specific primers for RSVA are SEQ ID NO. 9-10, the specific primers for RSVB are SEQ ID NO. 11-12, the specific primers for HRV are SEQ ID NO. 13-14, the specific primers for H3N2 are SEQ ID NO. 15-16, the specific primers for ADV3 are SEQ ID NO. 17-18, the specific primers for ADV7 are SEQ ID NO. 19-20, the specific primers for HMPV are SEQ ID NO. 21-22, and the specific primers for MP are SEQ ID NO. 23-24; the specific probe for 2019-nCoV is SEQ ID NO.27, the specific probe for 2019-nCoV is SEQ ID NO.28, the specific probe for FluA is SEQ ID NO.29, the specific probe for FluB is SEQ ID NO.30, the specific probe for RSVA is SEQ ID NO.31, the specific probe for RSVB is SEQ ID NO.32, the specific probe for HRV is SEQ ID NO.33, the specific probe for H3N2 is SEQ ID NO.34, the specific probe for ADV3 is SEQ ID NO.35, the specific probe for ADV7 is SEQ ID NO.36, the specific probe for HMPV is SEQ ID NO.37, and the specific probe for MP is SEQ ID NO. 38.
Preferably, in the technical scheme, the RT-PCR primer probe further comprises human ribonuclease P for human DNA internal reference, the specific primer of RNaseP is SEQ ID NO. 25-26, and the specific probe of RNaseP is SEQ ID NO. 39.
Preferably, in the above technical scheme, both ends of the specific probe are respectively provided with a fluorescent group and a quenching group.
Preferably, in the above technical solution, the detection method specifically includes the following steps: collecting a virus sample solution through a throat swab or sputum, and extracting nucleic acid aiming at the virus sample solution; performing RT-PCR amplification reaction by using the extracted nucleic acid as a template and collecting fluorescence; and (4) interpretation of results: and (3) judging the negative and positive: the amplification curve is S-shaped, and the Ct value is less than or equal to 38, and the amplification curve is judged to be positive; the Ct value is greater than 38, and the result is judged to be negative; when the negative control detection is negative and the positive control detection is positive, if the sample to be detected is positive, the result is judged to be positive; when the negative control detection is negative and the positive control detection is positive, if the sample to be detected is negative, the result is judged to be virus nucleic acid negative; and (3) judging the virus type: detecting a positive sample aiming at the virus nucleic acid, and judging that the sample is 2019-nCoV positive when the peak value of a first channel melting curve is at 55.1 +/-1.0 ℃ and 62 +/-1.0 ℃; when the peak value of the first channel melting curve is 70.8 +/-1.0 ℃, judging that the FluA is positive; when the peak value of the first channel melting curve is 77 +/-1.0 ℃, judging that the FluB is positive; when the peak value of the second channel melting curve is 57.5 +/-1.0 ℃, the RSVA is judged to be positive; when the peak value of the second channel melting curve is 64 +/-1.0 ℃, judging that the second channel melting curve is RSVB positive; when the peak value of the second channel melting curve is 70.2 +/-1.0 ℃, the HRV is judged to be positive; when the peak value of the second channel melting curve is 78 +/-1.0 ℃, judging that the second channel melting curve is positive H3N 2; when the peak value of the melting curve of the third channel is 54.9 +/-1.0 ℃, the HAdV3 is judged to be positive; when the peak value of the melting curve of the third channel is 62.3 +/-1.0 ℃, the HAdV7 is judged to be positive; when the peak value of the melting curve of the third channel is 69.7 +/-1.0 ℃, determining that the HMPV is positive; when the peak value of the melting curve of the third channel is 77 +/-1.0 ℃, determining that the MP is positive; when the peak value of the melting curve is out of all the ranges, the detection fails, and the detection needs to be repeated; wherein, the RT-PCR reaction system contains sample nucleic acid, enzyme mixed liquor and RT-PCR primer probe; RT-PCR amplification reactions RT-PCR amplifications were performed according to the following procedure: 15min at 50 ℃; 5min at 95 ℃; at 95 ℃ for 10s and at 58 ℃ for 40s, and carrying out 45 cycles; melting curve analysis was performed according to the following procedure: 10s at 95 ℃; 1min at 40 ℃; starting to run a melting curve analysis program from 40-85 ℃;
preferably, in the above technical solution, the detection solution comprises PCR buffer solution and 3.3mM MgCl20.1-0.4mM dNTPs (dATP: dTTP: dCTP: dGTP: dUTP =1:1:1:1: 1), primer probes (SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13, SEQ ID No.15, SEQ ID No.17, SEQ ID No.19, SEQ ID No.21, SEQ ID No.23, SEQ ID No.25, SEQ ID No.26 at a concentration of 0.4. mu.M; SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID No.10, SEQ ID No.12, SEQ ID No.14, SEQ ID No.16, SEQ ID No.18, SEQ ID No.20, SEQ ID No.22, SEQ ID No.24 at a concentration of 0.04. mu.M; SEQ ID No.27, 0.39. mu.M) and a reverse transcriptase inhibitor, wherein the enzyme concentration is selected from the group consisting of DNA polymerase, the enzyme and the enzyme mixture of the above DNA polymerase, the enzyme and the reverse transcriptase inhibitor, the enzyme mixture; the positive control is an artificially synthesized gene, and the negative control is an artificially synthesized gene containing an RNaseP gene fragment.
Compared with the prior art, the invention has the following beneficial effects:
the probe and the primer composition for rapidly detecting various respiratory pathogens adopt the technology of combining asymmetric multiplex fluorescence quantitative PCR and a melting curve, respectively design specific molecular beacons or Taqman probes according to different target genes, and particularly carry out multi-target simultaneous detection on 2019-nCoV, so that the detection specificity is ensured, and the occurrence of false positive is avoided. Meanwhile, the internal standard is arranged, so that false negative of a detection result can be effectively avoided, and the collection, transportation and extraction processes of a detection sample are monitored. In addition, according to the combination of different gene amplification fragments and fluorescent probes, melting curve peaks at different positions are generated in different fluorescent channels, so that the simultaneous detection of multiple pathogens by a single tube can be quickly and effectively realized, and the detection flux is greatly improved.
Description of the drawings:
FIG. 1 is a schematic view of the melting curves of 2019-nCoV, FluA and FluB types.
FIG. 2 is a schematic view of melting curves of RSVA, RSVB, HRV, H3N2 types.
FIG. 3 is a schematic view showing the melting curves of the types HAdV3, HAdV7, HMPV and MP.
The specific implementation mode is as follows:
the following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments.
Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.
EXAMPLE 1 design of specific primers and probes
Downloading ORF1ab and N gene of 2019-nCoV through NCBI search; FluA, M gene of MPV; the N gene of FluB; p genes of RSVA and RSVB; the 5' UTR gene of HRV; the HA gene of H3N 2; HEXON genes of HAdV3 and HAdV 7; MP CARDS gene and RNaseP gene were synthesized by Shanghai Shuoyao Biotechnology, Inc. by designing specific amplification primers and molecular beacons or Taqman probes for the above pathogens, respectively, using Beacon Designer or Primer 3 software. The primers and probes used for detection are shown in table 1:
TABLE 1 primer and Probe sequence information
Figure RE-RE-DEST_PATH_IMAGE001
Example 2 sample nucleic acid extraction
(1) Collecting, storing and transporting clinical samples: is suitable for throat swab and sputum sample types. Throat swab: the swab is passed over the root of the tongue, the two pharyngeal tonsils of the person to be collected are wiped back and forth slightly and forcefully for at least 3 times, then the posterior pharyngeal wall is wiped up and down for at least 3 times, the swab head is immersed into a tube containing virus preservation solution (isotonic saline solution, tissue culture solution or phosphate buffer solution can also be used), the tail part is discarded, and the tube cover is screwed tightly. Sputum: the expectorated sputum was collected in 50 mL screw plastic tubes or sputum boxes containing viral stocks. Adding sputum digestive juice (phosphate buffer solution containing 1g/L proteinase K) with the same volume into the sputum sample, shaking and uniformly mixing, standing for 5 minutes for digestion, and then carrying out nucleic acid extraction. The digested sputum sample can be used as a conventional swab sample for subsequent processing. Samples for virus separation and nucleic acid detection should be detected as soon as possible, and samples capable of being detected within 24 hours can be stored at 4 ℃; samples which can not be detected within 24 hours are stored at the temperature of-70 ℃ or below (if the storage condition of-70 ℃ is not available, the samples are stored in a refrigerator at the temperature of-20 ℃). The freezing and thawing of the sample can not exceed 5 times, otherwise, the detection result is influenced.
(2) Nucleic acid extraction: the QIAamp Viral RNA Mini Kit from Qiagen company is adopted for extracting the sample nucleic acid, and the nucleic acid extraction is carried out according to the instruction.
Example 3 detection of extracted sample nucleic acid
The amplification reaction system was prepared as shown in Table 2:
TABLE 2 amplification reaction System preparation Table
Figure RE-RE-DEST_PATH_IMAGE003
RT-PCR amplification was performed according to the following procedure: 15min at 50 ℃; 5min at 95 ℃; at 95 ℃ for 10s and at 58 ℃ for 40s, and carrying out 45 cycles;
melting curve analysis was performed according to the following procedure: 10s at 95 ℃; 1min at 40 ℃; the melting curve analysis program was run starting from 40-85 ℃.
Example 4 analysis of results
Determining whether the respiratory tract pathogen to be detected exists in the sample according to the melting curve peak diagram and the internal standard amplification condition, and judging the type of the infected virus according to the type and the melting temperature of the fluorescence channel, wherein the melting temperature corresponding to each type of virus is shown in a table 3:
TABLE 3 interpretation of results
Figure RE-DEST_PATH_IMAGE004
Example 5 specific assay
The method of the invention is adopted to treat coronavirus 229E, coronavirus OC43, coronavirus HKU1, coronavirus NL63, parainfluenza virus (1, 2 and 3), bocavirus, enterovirus (EV 71, echovirus 30, poliovirus 1 and EV-D68), EB virus, measles virus, human cytomegalovirus, rotavirus (group A), norovirus (group G II), mumps virus, varicella-zoster virus and chlamydia pneumoniae, legionella, pertussis, haemophilus influenzae, staphylococcus aureus, streptococcus pneumoniae, streptococcus pyogenes, klebsiella pneumoniae, mycobacterium tuberculosis, aspergillus fumigatus, candida albicans, candida glabrata, cryptococcus neoformans, neisseria meningitidis, streptococcus salivarius, and human leukocyte total nucleic acid were subjected to multiplex fluorescent quantitative PCR amplification and melting curve analysis.
Example 6 sensitivity analysis
Diluting the artificially synthesized gene containing each virus target fragment according to 10-fold gradient, performing multiplex fluorescence quantitative PCR amplification and melting curve analysis, and analyzing the detection minimum limit of 5 × 102copies/mL。
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
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<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
acttamgmag caracgtgac a 21
<210> 16
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ggctaccaac tagtgtgcta ag 22
<210> 17
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
cctagtccat ggcaatctak tta 23
<210> 18
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
tttacaaggt ccagacgttc at 22
<210> 19
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
aatccctatg cataagctgc a 21
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
gaaagggctc agtacaggct 20
<210> 21
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
cttcartatc ctcagargca ag 22
<210> 22
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
atatcayygc acgtactgac ataa 24
<210> 23
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
aacaartctg gttaggctat aat 23
<210> 24
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
ccacgtagga atttgatacc rag 23
<210> 25
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
cacttgcatg acttaggaca gt 22
<210> 26
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
acgtttggca tgcggattac gg 22
<210> 27
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
tgcgtacgta ccagtcgcta ac 22
<210> 28
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
ccacgtaaca gtgcgtaagg t 21
<210> 29
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
tcttcgccca tacgcctaag atc 23
<210> 30
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
cacctgaact cgaatcgata agc 23
<210> 31
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
ttagccatag gccgttcata gcct 24
<210> 32
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
tcctcataac gccgtagaag ctac 24
<210> 33
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
tggactaaac gcggtatgcg act 23
<210> 34
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
atccgttatc aagccctaag cc 22
<210> 35
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
cagtacagcc tgaactgcac atc 23
<210> 36
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
atgccatggc gatcgattcg cac 23
<210> 37
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
tgcatggcag aaccgtcagc tg 22
<210> 38
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
tgtaggcatg cagacctagg cac 23
<210> 39
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
tggatcgtaa cgtcgatacg gct 23

Claims (10)

1. A kit for rapidly detecting multiple respiratory pathogens is characterized by comprising specific primers aiming at 2019-nCoV, FluA, FluB, H3N2, RSVA, RSVB, HRV, HAdV3, HAdV7, HMPV and MP, wherein the specific primers aiming at the N gene of 2019-nCoV are SEQ ID NO. 1-2, the specific primers aiming at ORF1ab gene of 2019-nCoV are SEQ ID NO. 3-4, the specific primers aiming at M gene of FluA are SEQ ID NO. 5-6, the specific primers aiming at N gene of FluB are SEQ ID NO. 7-8, the specific primers aiming at P gene of RSVA are SEQ ID NO. 9-10, the specific primers aiming at P gene of VB are SEQ ID NO. 11-12, the specific primers aiming at 5' gene of HRV are SEQ ID NO. 13-14, the specific primers aiming at HA gene N2 are SEQ ID NO. 15-3, and the specific primers aiming at ADV NO. 18-8617, the HEXON gene specific primers aiming at ADV7 are SEQ ID NO. 19-20, the M gene specific primers aiming at HMPV are SEQ ID NO. 21-22, and the CARDS gene specific primers aiming at MP are SEQ ID NO. 23-24.
2. The kit for rapidly detecting multiple respiratory pathogens according to claim 1, wherein the kit comprises: the primer also comprises specific primers of human ribonuclease P and RNaseP aiming at human DNA internal reference, wherein the specific primers are SEQ ID NO. 25-26.
3. A kit for rapidly detecting multiple respiratory pathogens, which is characterized by comprising specific probes for 2019-nCoV, FluA, FluB, H3N2, RSVA, RSVB, HRV, HAdV3, HAdV7, HMPV and MP, wherein the specific probe for the N gene of 2019-nCoV is SEQ ID NO.27, the specific probe for ORF1ab gene of 2019-nCoV is SEQ ID NO.28, the specific probe for the M gene of FluA is SEQ ID NO.29, the specific probe for the N gene of FluB is SEQ ID NO.30, the specific probe for the P gene of RSVA is SEQ ID NO.31, the specific probe for the P gene of RSVB is SEQ ID NO.32, the specific probe for the 5' UTR gene of HRV is SEQ ID NO.33, the specific probe for the HA gene of H3N2 is SEQ ID NO.34, the specific probe for the HEXON gene of ADV3 is SEQ ID NO.35, and the specific probe for the ADV7, the M gene specific probe for HMPV was SEQ ID No.37 and the CARDS gene specific probe for MP was SEQ ID No. 38.
4. The kit for rapidly detecting multiple respiratory pathogens according to claim 3, wherein: also includes specific probe of RNaseP for human DNA reference SEQ ID NO. 39.
5. The kit for rapidly detecting multiple respiratory pathogens according to claim 3 or 4, wherein: the two ends of the specific probe are respectively provided with a fluorescent group and a quenching group, wherein the fluorescent group is selected from any one or more of FAM, HEX, VIC, TET, TAMRA, ROX, CY3.5 or CY 5; the quenching group is selected from any one or more of BHQ1, BHQ2, BHQ3, DABCYL and MGB.
6. A detection method for rapidly detecting various respiratory pathogens comprises the step of adding a sample to be detected into an RT-PCR reaction system containing an RT-PCR primer probe to perform real-time fluorescent quantitative RT-PCR reaction, and is characterized in that the RT-PCR primer probe comprises an amplification primer pair and a detection probe aiming at 2019-nCoV, FluA, FluB, H3N2, RSVA, RSVB, HRV, HAdV3, HAdV7, HMPV and MP respectively; wherein: the specific primers for 2019-nCoV are SEQ ID NO. 1-2, the specific primers for 2019-nCoV are SEQ ID NO. 3-4, the specific primers for FluA are SEQ ID NO. 5-6, the specific primers for FluB are SEQ ID NO. 7-8, the specific primers for RSVA are SEQ ID NO. 9-10, the specific primers for RSVB are SEQ ID NO. 11-12, the specific primers for HRV are SEQ ID NO. 13-14, the specific primers for H3N2 are SEQ ID NO. 15-16, the specific primers for ADV3 are SEQ ID NO. 17-18, the specific primers for ADV7 are SEQ ID NO. 19-20, the specific primers for HMPV are SEQ ID NO. 21-22, and the specific primers for MP are SEQ ID NO. 23-24; the specific probe for 2019-nCoV is SEQ ID NO.27, the specific probe for 2019-nCoV is SEQ ID NO.28, the specific probe for FluA is SEQ ID NO.29, the specific probe for FluB is SEQ ID NO.30, the specific probe for RSVA is SEQ ID NO.31, the specific probe for RSVB is SEQ ID NO.32, the specific probe for HRV is SEQ ID NO.33, the specific probe for H3N2 is SEQ ID NO.34, the specific probe for ADV3 is SEQ ID NO.35, the specific probe for ADV7 is SEQ ID NO.36, the specific probe for HMPV is SEQ ID NO.37, and the specific probe for MP is SEQ ID NO. 38.
7. The assay for the rapid detection of multiple respiratory pathogens according to claim 6 wherein: the RT-PCR primer probe also comprises human ribonuclease P aiming at human DNA internal reference, the specific primer of RNaseP is SEQ ID NO. 25-26, and the specific probe of RNaseP is SEQ ID NO. 39.
8. The method for rapidly detecting multiple respiratory pathogens according to claim 6 or 7, wherein the method comprises the following steps: the two ends of the specific probe are respectively provided with a fluorescent group and a quenching group.
9. The detection method for rapidly detecting multiple respiratory pathogens according to claim 6, wherein the detection method comprises the following steps: collecting a virus sample solution through a throat swab or sputum, and extracting nucleic acid aiming at the virus sample solution; performing RT-PCR amplification reaction by using the extracted nucleic acid as a template and collecting fluorescence; and (4) interpretation of results: and (3) judging the negative and positive: the amplification curve is S-shaped, and the Ct value is less than or equal to 38, and the amplification curve is judged to be positive; the Ct value is greater than 38, and the result is judged to be negative; when the negative control detection is negative and the positive control detection is positive, if the sample to be detected is positive, the result is judged to be positive; when the negative control detection is negative and the positive control detection is positive, if the sample to be detected is negative, the result is judged to be virus nucleic acid negative; and (3) judging the virus type: detecting a positive sample aiming at the virus nucleic acid, and judging that the sample is 2019-nCoV positive when the peak value of a first channel melting curve is at 55.1 +/-1.0 ℃ and 62 +/-1.0 ℃; when the peak value of the first channel melting curve is 70.8 +/-1.0 ℃, judging that the FluA is positive; when the peak value of the first channel melting curve is 77 +/-1.0 ℃, judging that the FluB is positive; when the peak value of the second channel melting curve is 57.5 +/-1.0 ℃, the RSVA is judged to be positive; when the peak value of the second channel melting curve is 64 +/-1.0 ℃, judging that the second channel melting curve is RSVB positive; when the peak value of the second channel melting curve is 70.2 +/-1.0 ℃, the HRV is judged to be positive; when the peak value of the second channel melting curve is 78 +/-1.0 ℃, judging that the second channel melting curve is positive H3N 2; when the peak value of the melting curve of the third channel is 54.9 +/-1.0 ℃, the HAdV3 is judged to be positive; when the peak value of the melting curve of the third channel is 62.3 +/-1.0 ℃, the third channel is judged to be HAdV7 positive; when the peak value of the melting curve of the third channel is 69.7 +/-1.0 ℃, determining that the HMPV is positive; when the peak value of the melting curve of the third channel is 77 +/-1.0 ℃, determining that the MP is positive; when the peak value of the melting curve is out of all the ranges, the detection fails, and the detection needs to be repeated; wherein, the RT-PCR reaction system contains sample nucleic acid, enzyme mixed liquor and RT-PCR primer probe; RT-PCR amplification reactions RT-PCR amplifications were performed according to the following procedure: 15min at 50 ℃; 5min at 95 ℃; at 95 ℃ for 10s and at 58 ℃ for 40s, and performing 45 cycles; melting curve analysis was performed according to the following procedure: 10s at 95 ℃; 1min at 40 ℃; the melting curve analysis program was run starting from 40-85 ℃.
10. The method for rapid detection of multiple respiratory pathogens according to claim 9, wherein the detection solution comprises PCR buffer, 3.3mM MgCl20.1-0.4mM dNTPs (dATP: dTTP: dCTP: dGTP: dUTP =1:1:1:1: 1), primer probes(the concentrations of SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13, SEQ ID No.15, SEQ ID No.17, SEQ ID No.19, SEQ ID No.21, SEQ ID No.23, SEQ ID No.25 and SEQ ID No.26 are 0.4. mu.M; the concentrations of SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID No.10, SEQ ID No.12, SEQ ID No.14, SEQ ID No.16, SEQ ID No.18, SEQ ID No.20, SEQ ID No.22 and SEQ ID No.24 are 0.04. mu.M; the concentrations of SEQ ID Nos. 27 to 39 are all 0.2. mu.M) and a water for removing RNA enzyme, the enzyme mixture including an RNA enzyme inhibitor, DNA polymerase, reverse transcriptase; the positive control is an artificially synthesized gene, and the negative control is an artificially synthesized gene containing an RNaseP gene fragment.
CN202110733994.8A 2021-06-30 2021-06-30 Kit for rapidly detecting multiple respiratory pathogens, probe and primer combination Withdrawn CN114574630A (en)

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