CN113981143A - Kit for detecting 8 respiratory pathogens containing Xinguan and application thereof - Google Patents
Kit for detecting 8 respiratory pathogens containing Xinguan and application thereof Download PDFInfo
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Abstract
The invention discloses a detection kit containing 8 respiratory pathogens of Xinguan and application thereof, wherein the kit comprises specific amplification primers for amplifying 9 gene loci of the following 8 respiratory pathogens; wherein, the 9 gene loci are: 2019 ORF and N gene of novel coronavirus, hemagglutinin gene of influenza A virus, M protein gene of influenza B virus, upstream segment of Hexon gene Loop1 of adenovirus, hemagglutinin-neuraminidase gene of parainfluenza virus, F protein gene of respiratory syncytial virus, VP1 capsid protein gene of rhinovirus, 16S rRNA gene of mycoplasma pneumoniae; and specific amplification primers of the reference gene ACTB. The kit adopts a multiple RT-qPCR technology, takes the highly conserved sequences of the human endogenous reference gene, the novel coronavirus SARS-CoV-2 and other 7 common respiratory viruses as target regions, designs 9 groups of specific primers and probes, constructs a 3-tube quadruple detection system, and has higher specificity.
Description
Technical Field
The invention belongs to the technical field of molecular diagnosis, and relates to a detection kit covering novel coronavirus and other 7 respiratory pathogens, in particular to a detection kit containing 8 respiratory pathogens of new corona and application thereof.
Background
The initial symptoms of the novel coronavirus (SARS-CoV-2) infection mainly comprise fever, general pain, hypodynamia and the like, and are similar to the symptoms of other respiratory pathogens such as influenza A virus (inflenzaa), influenza B virus (inflenzab), adenovirus (HAdv), parainfluenza virus (HPIV), respiratory syncytial virus (HRSV) and the like.
The current detection method for diagnosing respiratory virus infection in China mainly comprises the following steps: 1) a culture method; 2) mass spectrometry; 3) immunological detection; 4) PCR method, etc. Among them, the living body from which a pathogen is obtained (mainly culture positive for bacteria, fungi, and viruses) is a gold standard for diagnosis of infectious diseases. However, in vitro culture often shows negative results in the early stage of infection, the time consumption of in vitro culture of pathogens is generally long, the operation steps are complicated, most viruses are difficult to culture and identify, and the detection rate is low. The immunological method, such as neutralization test, enzyme-linked immunosorbent assay, immunofluorescence method, enzyme-labeled spot immunoassay, etc., is simple to operate, but because of the various pathogen types, the quantity of the developed antigen and antibody can not meet the clinical requirement. Mass spectrometry has the advantages of high efficiency and sensitivity, but detection depends on expensive instruments and equipment, and extremely professional operators are needed, so that mass popularization and application are difficult. With the progress of molecular biology, the development of PCR technology provides a new choice for the identification and typing of pathogens. However, for unknown pathogen infection and multi-pathogen mixed infection, the conventional PCR method usually requires multiple amplification screens, which is relatively time-consuming and labor-consuming. The multiplex qPCR method has the characteristics of high sensitivity and high specificity, has obvious advantages in the detection of unknown pathogen infection and multi-pathogen mixed infection, and is a mature pathogen detection means at present.
At present, the kit products for multiplex detection of the respiratory viruses in China are few, and a Boo crystal core eight respiratory virus nucleic acid detection kit (constant temperature amplification chip method), a middle biological three respiratory virus nucleic acid detection kit (double amplification method) and a seven respiratory pathogen nucleic acid detection kit are sold on the market, but the products do not contain a novel coronavirus (SARS-CoV-2) detection target. There is no nucleic acid detection kit for simultaneous detection of SARS-CoV-2 and other various respiratory viruses.
Disclosure of Invention
The technical problem to be solved is as follows: in order to overcome the defects of the prior art, a method capable of simultaneously detecting novel coronavirus SARS-CoV-2 and other different respiratory viruses is obtained, namely: eight pathogens, namely 2019 novel coronavirus (SARS-CoV-2), influenza A virus (InfluenzaA, InfluenzaA H1N12009, InfluenzaA H3N2), influenza B virus (InfluenzaB), adenovirus (HAdV), parainfluenza virus (HPIV), respiratory syncytial virus (HRSV), rhinovirus (HRV) and Mycoplasma Pneumoniae (MP), can be qualitatively analyzed. In view of the above, the present invention provides a detection kit for 8 respiratory pathogens comprising neocorona and applications thereof.
The technical scheme is as follows: a kit for detecting 8 respiratory pathogens comprising neocorona, said kit comprising specific amplification primers for amplifying 9 genetic loci of the following 8 respiratory pathogens; wherein, the 9 gene loci are: 2019 ORF and N gene of novel coronavirus, hemagglutinin gene of influenza A virus, M protein gene of influenza B virus, upstream segment of Hexon gene Loop1 of adenovirus, hemagglutinin-neuraminidase gene of parainfluenza virus, F protein gene of respiratory syncytial virus, VP1 capsid protein gene of rhinovirus, 16S rRNA gene of mycoplasma pneumoniae; and specific amplification primers of the reference gene ACTB.
Preferably, the sequence of the specific amplification primer is as follows: SARS-CoV-2-ORF, SEQ ID NO. 1-2; SARS-CoV-2-N, SEQ ID NO. 4-5; HRSV, SEQ ID NO. 7-8; InFA, SEQ ID NO.10-11, InFB, SEQ ID NO. 13-14; HPIV, SEQ ID NO. 16-17; HAdV, SEQ ID NO. 19-20; HRV, SEQ ID NO. 22-23; MP, SEQ ID NO. 25-26; ACTB, SEQ ID NO. 28-29.
Preferably, the specific amplification primers are used at the final concentrations: SARS-CoV-2-ORF: 0.32. mu.M, SARS-CoV-2-N: 0.4 μ M, HRSV: 0.3. mu.M, InFA: 0.3 μ M, InFB: 0.4. mu.M, HPIV: 0.3. mu.M, HAdV: 0.3 μ M, HRV: 0.35. mu.M, MP: 0.32 μ M, ACTB: 0.4. mu.M.
Preferably, 1 degenerate probe is designed for 9 gene loci of the 8 respiratory pathogens and the reference gene ACTB, and the probe sequences are as follows: SARS-CoV-2-ORF, SEQ ID NO. 3; SARS-CoV-2-N, SEQ ID NO. 6; HRSV, SEQ ID NO. 9; InFA, SEQ ID NO.12, InFB, SEQ ID NO. 15; HPIV, SEQ ID NO. 18; HAdV, SEQ ID NO. 21; HRV, SEQ ID NO. 24; MP, SEQ ID NO. 27; ACTB, SEQ ID NO. 30.
Preferably, the probes are used at final concentrations of: SARS-CoV-2-ORF: 0.24. mu.M, SARS-CoV-2-N: 0.2 μ M, HRSV: 0.2. mu.M, InFA: 0.1 μ M, InFB: 0.3 μ M, HPIV: 0.3. mu.M, HAdV: 0.2 μ M, HRV: 0.25. mu.M, MP: 0.25 μ M, ACTB: 0.4. mu.M.
TABLE 1 sequences and concentrations of specific amplification primers and probes
TABLE 2 combinatorial design of 8 respiratory pathogen specific amplification primers comprising neocorona
TABLE 3 final concentrations used of combination A specific amplification primers
TABLE 4 final concentrations used of combination B specific amplification primers
TABLE 5 final concentrations used of combination C-specific amplification primers
Preferably, the probe is designed by using a TaqMan probe, wherein SARS-CoV-2-ORF, InFA and HAdV are fluorescently labeled by using FAM, and BHQ1 is selected as a quenching group; HEX fluorescence labeling is adopted for SARS-CoV-2-N, InFB and HRV, and BHQ1 is selected as a quenching group; HRSV, HPIV and MP are marked by ROX fluorescence, and BHQ2 is selected as a quenching group; ACTB was fluorescently labeled with Cy5 and the quenching group was BHQ 3.
Preferably, the 8 respiratory pathogens adopt a three-tube detection scheme, and specific amplification primers are specifically divided into the following 3 groups:
combination A: SARS-CoV-2-ORF, SARS-CoV-2-N, HRSV;
combination B: InFA, InFB, HPIV;
and (3) combination C: HAdV, HRV, MP;
and each combination contains the reference gene ACTB.
Among them, the primer sequences of influenza a viruses (inflenzaa, inflenzaa H1N12009, inflenzaa H3N2), parainfluenza virus (HPIV), respiratory syncytial virus (HRSV) and adenovirus (HAdV) can cover various subtypes, and thus the detection results are not classified.
The 8 respiratory tract pathogen detection kits containing the novel corona as described in any one of the above applications in the investigation of novel coronavirus infectors and the detection of upper respiratory tract pathogen infection.
Preferably, the detection sample of the kit is sputum, a pharyngeal swab or a nasal swab.
Has the advantages that: (1) the kit adopts a multiple RT-qPCR technology, takes the highly conserved sequences of human-derived internal reference genes, novel coronavirus SARS-CoV-2 and other 7 common respiratory viruses as target regions, designs 9 groups of specific primers and probes, and constructs a quadruple detection system with three channels; (2) compared with the approved new crown detection technology (qPCR, antibody), the kit has the advantages of multiple virus qualitative types, good specificity, more accurate qualitative, capability of reducing false negative probability, suitability for diagnosis of complex pathogen infection and capability of effectively avoiding missed diagnosis; (3) compared with the nucleic acid detection kit (constant temperature amplification chip method) for approved 6 respiratory viruses, the kit has the advantages that adenovirus and parainfluenza virus are added in the detection pathogen, the two viruses are common respiratory infection pathogens, and the prevalence rate in viral pneumonia is higher; the constant temperature amplification is easy to generate nonspecific reaction, false positive is caused, and the kit has higher specificity.
Drawings
FIG. 1 shows the amplification results of 2019 novel coronavirus (SARS-CoV-2) and respiratory syncytial virus (HRSV) detection target containing standard using the primer component of kit A according to some embodiments of the present application; as shown in fig. 1: 2019 the amplification Ct values of the novel coronavirus (SARS-CoV-2) and respiratory syncytial virus (HRSV) standard products are all within the positive judgment Ct value range (Ct is less than or equal to 38), which indicates that the detection result is positive.
FIG. 2 is a graph showing the amplification results of a standard containing influenza A virus (inflenza A, inflenza H1N12009, inflenza H3N2), influenza B virus (inflenza B), and parainfluenza virus (HPIV) detection targets using the primer set B of the kit according to some embodiments of the present disclosure; as shown in fig. 2: amplification Ct values of the standard products of influenza A viruses (Influenzaa, Influenzaa H1N12009 and Influenzaa H3N2), influenza B viruses (Influenzab) and parainfluenza viruses (HPIV) are all within a positive judgment Ct value range (Ct is less than or equal to 38), and the detection result is positive.
FIG. 3 is a graph showing the amplification of a standard containing adenovirus (HAdV), rhinovirus (HRV), Mycoplasma Pneumoniae (MP) detection targets using the primer set C of the kit according to some embodiments of the present disclosure; as shown in fig. 3: the amplification Ct values of the standard products of adenovirus (HAdV), rhinovirus (HRV) and Mycoplasma Pneumoniae (MP) are all within the positive judgment Ct value range (Ct is less than or equal to 38), which indicates that the detection result is positive.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and substance of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1: selection of target sequences and primer Probe design
For the selection of target sequences, the 2019 novel coronavirus (SARS-CoV-2) selects the common ORF1ab and N gene as its detection target. Influenza a viruses (inflenzaa), influenza a viruses H1N1(2009) (inflenzaah 1N12009), influenza a viruses H3N2 (inflenzaah 3N2) employ universal primer and probe design, selecting widely used hemagglutinin gene design primers and probes. Influenza b virus (influenza b) selects the M protein gene. For adenovirus (HAdv), 104 subtypes (http:// hadvwg. gmu. edu /), among which human adenovirus associated with respiratory disease mainly belong to subgenera B (HAdV-3, 7, 11, 14, 16, 21, 50, 55), subgenera C (HAdV-1, 2, 5, 6, 57) and subgenera E (HAdV-4) (see: human adenovirus respiratory infection prevention and control technical guidelines for (2019 edition)), the primer and probe design of the kit mainly belong to HAdV-3 and HAdV-7, and the design of the kit mainly belong to HAdV-11, HAdV-14, HAdV-55, HAdV-1, HAdV-2, HAdV-5 and HAdV-4, etc., and the Hexon late stage and Fiber genes of adenovirus are located on the transcription genes L3 and L5, respectively, are the main regions for typing, and the upstream region of Hexon gene of adenovirus is identified by the current technology (1), designing adenovirus general detection primer and probe. Parainfluenza virus (HPIV) has four main subtypes, and the embodiment adopts a non-typing mode, selects hemagglutinin-neuraminidase gene as a target point, and designs a universal detection primer and a probe. Human Respiratory Syncytial Virus (RSV) has only 1 serotype, two gene subtypes of A and B, the genome length is 15200bp, 11 proteins are coded, including 9 structural proteins such as adhesion protein (G), small hydrophobic protein (SH), fusion protein (F), nucleocapsid protein (N), phosphoprotein (P), polymerase and matrix protein (M1, M2-1, M2-2) and 2 non-structural proteins NS1 and NS2, wherein F, G and N proteins are main target antigens for vaccine research, and the F protein gene is selected as a detection target. The HRV-5' NCR (Non Coding Region, NCR) fragment of rhinovirus has relatively high conservation, and therefore, it was selected as the gene of interest. In the MP nucleic acid detection, primers and probes are usually designed with the ATPase operon gene, P1 adhesin gene, Repmpl and CARDS genes, and 16S rRNA gene of mycoplasma pneumoniae as targets, and since the 16S rRNA gene is the target, it has the advantage of high sensitivity, i.e. in the reverse transcription system, the template amount of the system can be increased by reverse transcription of 16S rRNA, thereby increasing the detection sensitivity, and also has the function of indicating whether active viruses exist in the sample, the present embodiment uses the 16S rRNA gene as the target, and designs specific primers and probes.
On the basis of determining a target sequence, a target spot detection Primer and a probe are designed by using a Primer Express 3.0, a Primer 5.0, an Oligo 7Primer Analysis and an NCBI online Analysis tool, and are submitted to synthesis.
In the design process of the PCR primer, the following factors are mainly considered: i.e., 19-28bp in length; the Tm values of the primers at each locus are as consistent as possible (about 60 ℃), and the calculation results of the Tm values are calculated according to Primer 5.0 software; the design of parameters is strict as much as possible, the formation of structures such as primer dimers, hairpins and the like is avoided as much as possible, the 3' end of a primer pair does not have complementary pairing of more than 3 bases, and the homology with other part sequences in the sequences is considered; the length of the PCR fragment is designed to be in a range of 50-150 bp; the probe mainly adopts a common TaqMan probe, FAM, HEX, ROX and Cy5 fluorescent dyes with high adaptability to an instrument are selected, BHQ series groups are selected as quenching groups, and the Tm value of the probe is controlled within the range of 65-70 ℃. Primer probes were synthesized and purified (HPLC grade) by shanghai bailey biotechnology limited; purity greater than 99% was confirmed by HPLC analysis; A260/A280 is greater than 1.2. Specific results are shown in table 1.
Example 2: pseudovirus Synthesis and evaluation
Synthesizing target gene segments of eight respiratory pathogens such as Xinguan by adopting an artificial synthesis method, constructing an MS2 expression vector, carrying out induced expression, carrying out concentration and purity identification after assembly and purification, and synthesizing pseudovirus by submitting Xiamen-Shangshan company Limited.
The concentration of the pseudovirus is verified after synthesis, and the concentration is not lower than 1010copies/mL, DNA residual less than 100 copies/mL.
Example 3: establishment of quadruple multiplex amplification system
Selecting RT-qPCR Mix of German-American biotechnology Limited in Wuxi, and carrying out performance evaluation (specificity and sensitivity) on the eight designed and synthesized pathogen detection primers such as Xinguan, preferably selecting the primer with the best performance for the construction of a subsequent composite primer system, and selecting the synthesized pseudovirus as a composite primer construction template.
After the single amplification performance evaluation is completed, primers and probes of different pathogens without mutual influence are selected for combined amplification, and the optimal pathogen combination collocation or the optimal primer and probe combination collocation is determined so as to realize the optimal re-amplification condition (namely the single amplification performance is consistent with the performance under the complex amplification), thereby completing the construction of a composite primer system. The concentration of the composite templates tends to be consistent, and the consistency of the amplification performance among primers is kept. Specific results are shown in tables 2 to 5.
Example 4: positive and negative standard substance coincidence rate
Detecting 10 negative and 10 positive standard substances, and the coincidence rate reaches more than 95%.
Example 5: sensitivity of detection
The lowest limit of detection LOD value is determined to be 200copies/mL, the detection is carried out for 20 times, and the positive coincidence rate reaches more than 95 percent.
Example 6: specificity of the kit
The test is repeated three times by using other samples (respiratory viruses, coronaviruses and the like) at the same position, and the coincidence rate is 100 percent.
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<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gaccatgtgg gcattacgtt taa 23
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
attgtgaatc ggctgactca ag 22
<210> 3
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ccgtgtgcag tatgagcaaa ggttacgg 28
<210> 4
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
agcgttgttc ggtatgtcg 19
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
caatttgatg ccacgtgtgt tg 22
<210> 6
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tcacacgttc gtgaccgtgg ttgt 24
<210> 7
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tatgcrctcg gtgtagttyg a 21
<210> 8
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
actttgggtg agrttggctc mtc 23
<210> 9
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
aaacaatcag catgtcttgc ratgygc 27
<210> 10
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
tgactaagcr gattctagga tatgtg 26
<210> 11
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
tacagtatga ctcckctgtc gat 23
<210> 12
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tcaccgtgcc cagtgagcga g 21
<210> 13
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
taacgctgct tgcacgtagt at 22
<210> 14
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
cctagatgag gtatgttgtc gta 23
<210> 15
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
ttaggacctc tgtggcaagc acagt 25
<210> 16
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ttgtggttca gacgygagag rtg 23
<210> 17
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
gctcctaaac atgamggata cc 22
<210> 18
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
agacaactga taggataytt ggaagt 26
<210> 19
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
agcatgcttc rgagtacctg ag 22
<210> 20
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
ccgcatactg aagtaghtgt ctgt 24
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
cgggcgract gcaycagacc 20
<210> 22
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
cccctgaatg yggctaacc 19
<210> 23
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
aaggaaaaag tgaaacacgg ac 22
<210> 24
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
cccaaagtag ttggtyccgt c 21
<210> 25
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
aagaactttg gttcgcayga atc 23
<210> 26
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
cttggtaggc cgttaccmc 19
<210> 27
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
agggttcgtt atttgatgag ggtgcg 26
<210> 28
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
cctggcaccc agcacaat 18
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
gccgatccac acggagtact 20
<210> 30
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
atcaagatca ttgctcctcc tgagcgc 27
Claims (9)
1. The kit for detecting 8 respiratory pathogens containing Xinguan is characterized by comprising specific amplification primers for amplifying 9 gene loci of the following 8 respiratory pathogens; wherein, the 9 gene loci are: 2019 ORF and N gene of novel coronavirus, hemagglutinin gene of influenza A virus, M protein gene of influenza B virus, upstream segment of Hexon gene Loop1 of adenovirus, hemagglutinin-neuraminidase gene of parainfluenza virus, F protein gene of respiratory syncytial virus, VP1 capsid protein gene of rhinovirus, 16S rRNA gene of mycoplasma pneumoniae; and specific amplification primers of the reference gene ACTB.
2. The kit for detecting 8 respiratory pathogens comprising neocorona according to claim 1, wherein the specific amplification primers have the following sequences: SARS-CoV-2-ORF, SEQ ID NO. 1-2; SARS-CoV-2-N, SEQ ID NO. 4-5; HRSV, SEQ ID NO. 7-8; InFA, SEQ ID NO.10-11, InFB, SEQ ID NO. 13-14; HPIV, SEQ ID NO. 16-17; HAdV, SEQ ID NO. 19-20; HRV, SEQ ID NO. 22-23; MP, SEQ ID NO. 25-26; ACTB, SEQ ID NO. 28-29.
3. The kit for detecting 8 respiratory pathogens comprising neocorona according to claim 2, wherein the specific amplification primers are used at final concentrations of: SARS-CoV-2-ORF: 0.32. mu.M, SARS-CoV-2-N: 0.4 μ M, HRSV: 0.3. mu.M, InFA: 0.3 μ M, InFB: 0.4. mu.M, HPIV: 0.3. mu.M, HAdV: 0.3 μ M, HRV: 0.35. mu.M, MP: 0.32 μ M, ACTB: 0.4. mu.M.
4. The kit for detecting 8 respiratory pathogens containing neocorona according to claim 1, wherein 1 degenerate probe is designed for each of 9 gene loci of the 8 respiratory pathogens and an internal reference gene ACTB, and the probe sequences are as follows: SARS-CoV-2-ORF, SEQ ID NO. 3; SARS-CoV-2-N, SEQ ID NO. 6; HRSV, SEQ ID NO. 9; InFA, SEQ ID NO.12, InFB, SEQ ID NO. 15; HPIV, SEQ ID NO. 18; HAdV, SEQ ID NO. 21; HRV, SEQ ID NO. 24; MP, SEQ ID NO. 27; ACTB, SEQ ID NO. 30.
5. The kit for detecting 8 respiratory pathogens comprising neocorona according to claim 4, wherein the probe is used at a final concentration of: SARS-CoV-2-ORF: 0.24. mu.M, SARS-CoV-2-N: 0.2 μ M, HRSV: 0.2. mu.M, InFA: 0.1 μ M, InFB: 0.3 μ M, HPIV: 0.3. mu.M, HAdV: 0.2 μ M, HRV: 0.25. mu.M, MP: 0.25 μ M, ACTB: 0.4. mu.M.
6. The 8 respiratory pathogens detection kit containing neocorona according to claim 4, wherein the probe is designed by TaqMan probe, SARS-CoV-2-ORF, InFA, HAdV are marked by FAM fluorescence, and the quenching group is BHQ 1; HEX fluorescence labeling is adopted for SARS-CoV-2-N, InFB and HRV, and BHQ1 is selected as a quenching group; HRSV, HPIV and MP are marked by ROX fluorescence, and BHQ2 is selected as a quenching group; ACTB was fluorescently labeled with Cy5 and the quenching group was BHQ 3.
7. The kit for detecting 8 respiratory pathogens containing neocorona according to claim 1, wherein the 8 respiratory pathogens are detected by a three-tube detection scheme, and specific amplification primers are specifically divided into the following 3 groups:
combination A: SARS-CoV-2-ORF, SARS-CoV-2-N, HRSV;
combination B: InFA, InFB, HPIV;
and (3) combination C: HAdV, HRV, MP;
and each combination contains the reference gene ACTB.
8. Use of the 8 respiratory pathogen detection kits of any of claims 1-7 comprising a novel corona for the screening of patients infected with a novel coronavirus and the detection of upper respiratory pathogen infection.
9. The use of claim 8, wherein the test sample of the kit is sputum, a pharyngeal swab, or a nasal swab.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114921588A (en) * | 2022-04-22 | 2022-08-19 | 江苏先声医学诊断有限公司 | Targeting segment and primer set for rhinovirus detection in a sensory sample suitable for ONT sequencing platform and application thereof |
| CN115807128A (en) * | 2022-10-25 | 2023-03-17 | 生工生物工程（上海）股份有限公司 | Nucleic acid combination, kit and detection method for detecting respiratory tract pathogens |
| CN115838836A (en) * | 2022-11-14 | 2023-03-24 | 圣湘生物科技股份有限公司 | Composition, kit and method for joint detection of different types of viruses and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012100370A1 (en) * | 2011-01-26 | 2012-08-02 | 中国人民解放军军事医学科学院微生物流行病研究所 | Methods and kits for detecting pathogens of respiratory tract |
| CN109609692A (en) * | 2018-12-25 | 2019-04-12 | 北京卓诚惠生生物科技股份有限公司 | For detecting the nucleic acid reagent, kit, system and method for respiratory tract infection pathogen |
| CN111074001A (en) * | 2019-12-19 | 2020-04-28 | 苏州华益美生物科技有限公司 | 9 respiratory tract pathogen nucleic acid synergistic multiplex PCR detection |
| CN111910021A (en) * | 2020-08-10 | 2020-11-10 | 北京百康芯生物科技有限公司 | Micro-fluidic chip for detecting respiratory tract pathogen nucleic acid and detection method |
| CN112342315A (en) * | 2020-03-20 | 2021-02-09 | 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 |
-
2021
- 2021-11-08 CN CN202111311941.3A patent/CN113981143A/en not_active Withdrawn
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012100370A1 (en) * | 2011-01-26 | 2012-08-02 | 中国人民解放军军事医学科学院微生物流行病研究所 | Methods and kits for detecting pathogens of respiratory tract |
| CN109609692A (en) * | 2018-12-25 | 2019-04-12 | 北京卓诚惠生生物科技股份有限公司 | For detecting the nucleic acid reagent, kit, system and method for respiratory tract infection pathogen |
| CN111074001A (en) * | 2019-12-19 | 2020-04-28 | 苏州华益美生物科技有限公司 | 9 respiratory tract pathogen nucleic acid synergistic multiplex PCR detection |
| CN112342315A (en) * | 2020-03-20 | 2021-02-09 | 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 |
| CN111910021A (en) * | 2020-08-10 | 2020-11-10 | 北京百康芯生物科技有限公司 | Micro-fluidic chip for detecting respiratory tract pathogen nucleic acid and detection method |
Non-Patent Citations (2)
| Title |
|---|
| HSING-YI CHUNG等: "Novel dual multiplex real-time RT-PCR assays for the rapid detection of SARS-CoV-2, influenza A/B, and respiratory syncytial virus using the BD MAX open system", EMERG MICROBES INFECT, vol. 10, no. 1, pages 161 - 166 * |
| 邢婉丽等: "一款用于快速检测包括SARS-CoV-2在内的19种常见呼吸道病毒的高通量、多指标等温扩增平台", ENGINEERING, vol. 6, no. 10, pages 1130 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114921588A (en) * | 2022-04-22 | 2022-08-19 | 江苏先声医学诊断有限公司 | Targeting segment and primer set for rhinovirus detection in a sensory sample suitable for ONT sequencing platform and application thereof |
| CN114921588B (en) * | 2022-04-22 | 2024-05-24 | 江苏先声医学诊断有限公司 | Targeting segment and primer set for detecting rhinovirus in sensing sample suitable for ONT sequencing platform and application thereof |
| CN115807128A (en) * | 2022-10-25 | 2023-03-17 | 生工生物工程（上海）股份有限公司 | Nucleic acid combination, kit and detection method for detecting respiratory tract pathogens |
| CN115807128B (en) * | 2022-10-25 | 2024-03-01 | 生工生物工程（上海）股份有限公司 | Nucleic acid combination, kit and detection method for detecting respiratory tract pathogens |
| CN115838836A (en) * | 2022-11-14 | 2023-03-24 | 圣湘生物科技股份有限公司 | Composition, kit and method for joint detection of different types of viruses and application thereof |
| CN115838836B (en) * | 2022-11-14 | 2024-01-30 | 圣湘生物科技股份有限公司 | Composition, kit, method and application of different types of virus joint inspection |
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