CN110358865A - For detecting kit and its application of Respirovirus - Google Patents
For detecting kit and its application of Respirovirus Download PDFInfo
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- CN110358865A CN110358865A CN201910677694.5A CN201910677694A CN110358865A CN 110358865 A CN110358865 A CN 110358865A CN 201910677694 A CN201910677694 A CN 201910677694A CN 110358865 A CN110358865 A CN 110358865A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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Abstract
It is the present invention relates to gene engineering technology field, in particular to a kind of for detecting the primer combination of probe of Respirovirus, including 16 pairs of primers and corresponding 16 probes.The present invention also provides the kits for detecting Respirovirus, including the primer combination of probe for detecting Respirovirus.The present invention also provides the kits for detecting Respirovirus in the application for detecting or assisting the non-diagnostic purpose in detection Respirovirus.16 kinds of Respirovirus can be detected simultaneously using the kit provided by the present invention for detecting Respirovirus, realize Multiple detection, and high sensitivity is efficient and convenient, it can be achieved that carrying out batch detection to sample.
Description
Technical field
The present invention relates to gene engineering technology field, in particular to a kind of kit for detecting Respirovirus and its
Using.
Background technique
Respiratory tract infection is one of most common infectious diseases, and main pathogens include bacterium, virus, mycoplasma, clothing
Substance, fungi etc., wherein mainly based on viral infection.Childhood infection respiratory pathogen usually induces acute respiratory sense
Dye, acute respiratory infection are one of most important threats of children, are mainly in children, the elderly and immunologic hypofunction person, especially
It is the common disease of paediatrics, frequently-occurring disease, wherein more than 90% being caused by respiratory virus infection.
The clinical common virus that can lead to respiratory tract infection includes Respiratory Syncytial Virus(RSV) A (RSVA), respiratory syncystial
Viral B (RSVB), influenza virus A (FluA), influenza virus B (FluB), coronavirus (OC43), coronal disease
Poison (229E), coronavirus (NL63), human metapneumovirus (MPV), bocavirus (HBOV), enterovirus (HEV), parainfluenza virus
1 (HPIV1) of poison, parainfluenza virus 2 (HPIV2), parainfluenza virus 3 (HPIV3), parainfluenza virus 4 (HPIV4), rhinovirus
(HRV) and adenovirus (ADV) etc..Currently, clinically the goldstandard of viral diagnosis is still Virus culture method.But time-consuming for the method,
Sensibility is low, it is high to require experimental implementation.Virus antigen-antibody detection can be used as infection evidence, and detection is quick, but sensibility is low,
It is easy to appear false positive and false negative result.
In view of foregoing description, it would be highly desirable to have a high sensitivity, specificity is good, and experimental implementation is simple, and time-consuming short for detecting
The method of Respirovirus occurs.
Summary of the invention
The purpose of the present invention is to provide a kind of for detecting the primer combination of probe of Respirovirus.
The second object of the present invention is to provide a kind of kit for detecting Respirovirus and its application, using this
Kit can detect 16 kinds of Respirovirus simultaneously, realize Multiple detection, high sensitivity, it is efficient and convenient, it can be achieved that sample into
Row batch detection.
In order to realize these purposes and other advantages according to the present invention, provide a kind of for detecting Respirovirus
Primer combination of probe, the primer combination of probe include:
1) as polynucleotide sequence primer pair 1 as shown in SEQ ID NO.1 and SEQ ID NO.2 and by polynucleotides sequence
Arrange the probe 1 as shown in SEQ ID NO.33；
2) as polynucleotide sequence primer pair 2 as shown in SEQ ID NO.3 and SEQ ID NO.4 and by polynucleotides sequence
Arrange the probe 2 as shown in SEQ ID NO.34；
3) as polynucleotide sequence primer pair 3 as shown in SEQ ID NO.5 and SEQ ID NO.6 and by polynucleotides sequence
Arrange the probe 3 as shown in SEQ ID NO.35；
4) as polynucleotide sequence primer pair 4 as shown in SEQ ID NO.7 and SEQ ID NO.8 and by polynucleotides sequence
Arrange the probe 4 as shown in SEQ ID NO.36；
5) as polynucleotide sequence primer pair 5 as shown in SEQ ID NO.9 and SEQ ID NO.10 and by polynucleotides
Sequence probe 5 as shown in SEQ ID NO.37；
6) as polynucleotide sequence primer pair 6 as shown in SEQ ID NO.11 and SEQ ID NO.12 and by polynucleotides
Sequence probe 6 as shown in SEQ ID NO.38；
7) as polynucleotide sequence primer pair 7 as shown in SEQ ID NO.13 and SEQ ID NO.14 and by polynucleotides
Sequence probe 7 as shown in SEQ ID NO.39；
8) as polynucleotide sequence primer pair 8 as shown in SEQ ID NO.15 and SEQ ID NO.16 and by polynucleotides
Sequence probe 8 as shown in SEQ ID NO.40；
9) as polynucleotide sequence primer pair 9 as shown in SEQ ID NO.17 and SEQ ID NO.18 and by polynucleotides
Sequence probe 9 as shown in SEQ ID NO.41；
10) as polynucleotide sequence primer pair 10 as shown in SEQ ID NO.19 and SEQ ID NO.20 and by multicore glycosides
Acid sequence probe 10 as shown in SEQ ID NO.42；
11) as polynucleotide sequence primer pair 11 as shown in SEQ ID NO.21 and SEQ ID NO.22 and by multicore glycosides
Acid sequence probe 11 as shown in SEQ ID NO.43；
12) as polynucleotide sequence primer pair 12 as shown in SEQ ID NO.23 and SEQ ID NO.24 and by multicore glycosides
Acid sequence probe 12 as shown in SEQ ID NO.44；
13) as polynucleotide sequence primer pair 13 as shown in SEQ ID NO.25 and SEQ ID NO.26 and by multicore glycosides
Acid sequence probe 13 as shown in SEQ ID NO.45；
14) as polynucleotide sequence primer pair 14 as shown in SEQ ID NO.27 and SEQ ID NO.28 and by multicore glycosides
Acid sequence probe 14 as shown in SEQ ID NO.46；
15) as polynucleotide sequence primer pair 15 as shown in SEQ ID NO.29 and SEQ ID NO.30 and by multicore glycosides
Acid sequence probe 15 as shown in SEQ ID NO.47；
16) as polynucleotide sequence primer pair 16 as shown in SEQ ID NO.31 and SEQ ID NO.32 and by multicore glycosides
Acid sequence probe 16 as shown in SEQ ID NO.48.
Wherein,
Primer pair 1 is used for the amplification of real-time monitoring primer pair 1 for expanding RSVA, probe 1；
Primer pair 2 is used for the amplification of real-time monitoring primer pair 2 for expanding RSVB, probe 2；
Primer pair 3 is used for the amplification of real-time monitoring primer pair 3 for expanding FluA, probe 3；
Primer pair 4 is used for the amplification of real-time monitoring primer pair 4 for expanding FluB, probe 4；
Primer pair 5 is used for the amplification of real-time monitoring primer pair 5 for expanding HBOV, probe 5；
Primer pair 6 is used for the amplification of real-time monitoring primer pair 6 for expanding OC43, probe 6；
Primer pair 7 is used for the amplification of real-time monitoring primer pair 7 for expanding 229E, probe 7；
Primer pair 8 is used for the amplification of real-time monitoring primer pair 8 for expanding NL63, probe 8；
Primer pair 9 is used for the amplification of real-time monitoring primer pair 9 for expanding HPIV1, probe 9；
Primer pair 10 is used for the amplification of real-time monitoring primer pair 10 for expanding HPIV2, probe 10；
Primer pair 11 is used for the amplification of real-time monitoring primer pair 11 for expanding HPIV3, probe 11；
Primer pair 12 is used for the amplification of real-time monitoring primer pair 12 for expanding HPIV4, probe 12；
Primer pair 13 is used for the amplification of real-time monitoring primer pair 13 for expanding MPV, probe 13；
Primer pair 14 is used for the amplification of real-time monitoring primer pair 14 for expanding HEV, probe 14；
Primer pair 15 is used for the amplification of real-time monitoring primer pair 15 for expanding ADV, probe 15；
Primer pair 16 is used for the amplification of real-time monitoring primer pair 16 for expanding HRV, probe 16.
Preferably, 5 ' ends of the probe are marked with any one of fluorescent reporter group FAM, JOE, ROX or Cy5；
3 ' ends of the probe are marked with fluorescent quenching group BHQ1.
Preferably, including above-mentioned for detecting the primer combination of probe of Respirovirus.
It preferably, further include negative control, positive control, internal reference, reaction amplification liquid, mixed enzyme solution and DEPC water；
Wherein, mixed enzyme solution includes Taq DNA enzymatic, reverse transcriptase and RNase inhibitor；Negative control is DEPC water；Positive control is
The plasmid of extension increasing sequence comprising 16 kinds of Respirovirus, internal reference are source of people beta-globin.
Preferably, the Respirovirus be Respiratory Syncytial Virus(RSV) A, Respiratory Syncytial Virus(RSV) B, influenza disease
Malicious A, influenza virus B, Coronavirus OC43, coronavirus 229E, coronavirus N L63, human metapneumovirus, rich card disease
Poison, enterovirus, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, rhinovirus and adenovirus.
Kit for detecting Respirovirus is detecting or is assisting the non-diagnostic purpose in detection Respirovirus
Using.
Preferably, the kit is 20uL detecting or assisting the reaction system of detection Respirovirus, specifically:
Sample 2uL to be detected, 2 × PCR buffer 10uL, mixed enzyme solution 0.5uL, primer combination of probe liquid 2uL, DEPC water is mended extremely
20uL；
Wherein, final concentration of 100nM-1000nM of the primer pair in amplification system；End of the probe in amplification system is dense
Degree is 50nM-500nM.Such as: primer pair final concentration of 100nM, 200nM, 500nM, 800nM in amplification system or
1000nM etc.；Final concentration of 50nM, 100nM, 200nM, 300nM, 400nM, the 500nM etc. of probe in amplification system.
The beneficial effects of the present invention are:
Provided by the present invention for detect Respirovirus kit, solve various respiratory road virus simultaneously detection and
16 kinds of Respirovirus can be detected simultaneously and be identified to the problem of identification, and this method is easy to operate, detection is accurate sensitive, special
It is anisotropic good, quick.
Provided by the present invention for detecting the kit of Respirovirus, Multiple detection can be realized to sample, it can be to sample
This progress batch detection.
Detailed description of the invention
Fig. 1 is the sensitivity experiment of primed probe group, wherein Fig. 1 a is that homemade RSVA Respirovirus detects positive matter
Grain sample carries out (10 after ten times of gradient dilutions2copies/ul、103copies/ul、104Copies/ul and 105copies/
Ul), the amplification curve for carrying out amplification reaction and obtaining, the standard curve that the amplification curve of Fig. 1 b according to fig. 1a is drawn, wherein R2
Value is 0.999；
Fig. 2 is the sensitivity experiment of primed probe group, wherein Fig. 2 a is that homemade RSVB Respirovirus detects positive matter
Grain sample carries out (10 after ten times of gradient dilutions2copies/ul、103copies/ul、104Copies/ul and 105copies/
Ul), the amplification curve for carrying out amplification reaction and obtaining, Fig. 2 b according to fig. 2 a amplification curve draw standard curve, wherein R2
Value is 0.999；
Fig. 3 is the sensitivity experiment of primed probe group, wherein Fig. 3 a is that homemade FluA Respirovirus detects positive matter
Grain sample carries out (10 after ten times of gradient dilutions2copies/uL、103copies/uLl、104Copies/uL and 105copies/
UL), the amplification curve for carrying out amplification reaction and obtaining, the standard curve that Fig. 3 b is drawn according to the amplification curve of Fig. 3 a, wherein R2
Value is 0.995；
Fig. 4 is the sensitivity experiment of primed probe group, wherein Fig. 4 a is that homemade FluB Respirovirus detects positive matter
Grain sample carries out (10 after ten times of gradient dilutions2copies/ul、103copies/ul、104Copies/ul and 105copies/
Ul), the amplification curve for carrying out amplification reaction and obtaining, the standard curve that Fig. 4 b is drawn according to the amplification curve of Fig. 4 a, wherein R2
Value is 0.998；
Fig. 5 is the sensitivity experiment of primed probe group, wherein Fig. 5 a is that homemade HBOV Respirovirus detects positive matter
Grain sample carries out (10 after ten times of gradient dilutions2copies/ul、103copies/ul、104Copies/ul and 105copies/
Ul), the amplification curve for carrying out amplification reaction and obtaining, the standard curve that Fig. 5 b is drawn according to the amplification curve of Fig. 5 a, wherein R2
Value is 0.999；
Fig. 6 is the sensitivity experiment of primed probe group, wherein Fig. 6 a is that homemade OC43 Respirovirus detects positive matter
Grain sample carries out (10 after ten times of gradient dilutions2copies/ul、103copies/ul、104Copies/ul and 105copies/
Ul), the amplification curve for carrying out amplification reaction and obtaining, the standard curve that Fig. 6 b is drawn according to the amplification curve of Fig. 6 a, wherein R2
Value is 0.999；
Fig. 7 is the sensitivity experiment of primed probe group, wherein Fig. 7 a is that homemade 29E Respirovirus detects positive matter
Grain sample carries out (10 after ten times of gradient dilutions2copies/ul、103copies/ul、104Copies/ul and 105copies/
Ul), the amplification curve for carrying out amplification reaction and obtaining, the standard curve that Fig. 7 b is drawn according to the amplification curve of Fig. 7 a, wherein R2
Value is 0.998；
Fig. 8 is the sensitivity experiment of primed probe group, wherein Fig. 8 a is that homemade NL63 Respirovirus detects positive matter
Grain sample carries out (10 after ten times of gradient dilutions2copies/ul、103copies/ul、104Copies/ul and 105copies/
Ul), the amplification curve for carrying out amplification reaction and obtaining, the standard curve that Fig. 8 b is drawn according to the amplification curve of Fig. 8 a, wherein R2
Value is 0.922；
Fig. 9 is the sensitivity experiment of primed probe group, wherein Fig. 9 a is that the detection of homemade HPIV1 Respirovirus is positive
Plasmid samples carry out (10 after ten times of gradient dilutions2copies/ul、103copies/ul、104Copies/ul and 105copies/
Ul), the amplification curve for carrying out amplification reaction and obtaining, the standard curve that Fig. 9 b is drawn according to the amplification curve of Fig. 9 a, wherein R2
Value is 0.998；
Figure 10 is the sensitivity experiment of primed probe group, wherein Figure 10 a is homemade HPIV2 Respirovirus detection sun
Property Plasmid samples carry out ten times of gradient dilutions after (102copies/ul、103copies/ul、104Copies/ul and
105Copies/ul), the amplification curve for carrying out amplification reaction and obtaining, the standard that Figure 10 b is drawn according to the amplification curve of Figure 10 a
Curve, wherein R2Value is 0.995；
Figure 11 is the sensitivity experiment of primed probe group, wherein Figure 11 a is homemade HPIV3 Respirovirus detection sun
Property Plasmid samples carry out ten times of gradient dilutions after (102copies/ul、103copies/ul、104Copies/ul and
105Copies/ul), the amplification curve for carrying out amplification reaction and obtaining, the standard that Figure 11 b is drawn according to the amplification curve of Figure 11 a
Curve, wherein R2Value is 0.999；
Figure 12 is the sensitivity experiment of primed probe group, wherein Figure 12 a is homemade HPIV4 Respirovirus detection sun
Property Plasmid samples carry out ten times of gradient dilutions after (102copies/ul、103copies/ul、104Copies/ul and
105Copies/ul), the amplification curve for carrying out amplification reaction and obtaining, the standard that Figure 12 b is drawn according to the amplification curve of Figure 12 a
Curve, wherein R2Value is 0.998；
Figure 13 is the sensitivity experiment of primed probe group, wherein Figure 13 a is that the detection of homemade MPV Respirovirus is positive
Plasmid samples carry out (10 after ten times of gradient dilutions2copies/ul、103copies/ul、104Copies/ul and 105copies/
Ul), the amplification curve for carrying out amplification reaction and obtaining, the standard curve that Figure 13 b is drawn according to the amplification curve of Figure 13 a, wherein
R2Value is 0.999；
Figure 14 is the sensitivity experiment of primed probe group, wherein Figure 14 a is that the detection of homemade HEV Respirovirus is positive
Plasmid samples carry out (10 after ten times of gradient dilutions2copies/ul、103copies/ul、104Copies/ul and 105copies/
Ul), the amplification curve for carrying out amplification reaction and obtaining, the standard curve that Figure 14 b is drawn according to the amplification curve of Figure 14 a, wherein
R2Value is 0.985；
Figure 15 is the sensitivity experiment of primed probe group, wherein Figure 15 a is that the detection of homemade ADV Respirovirus is positive
Plasmid samples carry out (10 after ten times of gradient dilutions2copies/ul、103copies/ul、104Copies/ul and 105copies/
Ul), the amplification curve for carrying out amplification reaction and obtaining, the standard curve that Figure 15 b is drawn according to the amplification curve of Figure 15 a, wherein
R2Value is 0.972；
Figure 16 is the sensitivity experiment of primed probe group, wherein Figure 16 a is that the detection of homemade HRV Respirovirus is positive
Plasmid samples carry out (10 after ten times of gradient dilutions2copies/ul、103copies/ul、104Copies/ul and 105copies/
Ul), the amplification curve for carrying out amplification reaction and obtaining, the standard curve that Figure 16 b is drawn according to the amplification curve of Figure 16 a, wherein
R2Value is 0.998.
Specific embodiment
Present invention will be described in further detail below with reference to the accompanying drawings, to enable those skilled in the art referring to specification text
Word can be implemented accordingly.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein do not allot one or more
The presence or addition of a other elements or combinations thereof.
Embodiment 1
The detection of Respirovirus:
(1) extraction of virus genom DNA
Collect person's Nasopharyngeal swabs to be checked or Sputum samples using commercially available virus genom DNA/RNA extracts kit, specifically
Operating process carries out to specifications.
(2) design of primers
Respiratory Syncytial Virus(RSV) A (RSVA), Respiratory Syncytial Virus(RSV) virus B (RSVB), popularity are obtained from GenBank
Common cold virus A (FluA), influenza virus B (FluB), coronavirus (OC43), coronavirus (229E), coronavirus
(NL63), human metapneumovirus (MPV), bocavirus (HBOV), enterovirus (HEV), parainfluenza virus 1 (HPIV1), parainfluenza
2 (HPIV2) of virus, parainfluenza virus 3 (HPIV3), parainfluenza virus 4 (HPIV4), rhinovirus (HRV) and adenovirus (ADV)
Corresponding sequence, design amplimer (as shown in table 1), it is ensured that each pair of primer can amplify corresponding virus, not with it is other
Non-specific amplification occurs for virus.
1 16 kinds of Respirovirus amplimers of table and probe
(3) foundation of multiple PCR method
Using kit provided by the invention, reaction system 20uL, specifically: nucleic acid-templated (sample to be tested) 2uL,
2 × PCR buffer 10uL, mixed enzyme solution 0.5uL, primed probe mixed liquor 2uL, DEPC water is mended to 20uL.Wherein enzyme mixes
Liquid includes Taq enzyme (1U/ μ l), reverse transcriptase M-MLV (0.5U/ μ l), UNG enzyme (0.05U/ μ l).Nucleic acid amplification reaction liquid includes
Magnesium chloride (25mM), 10mMdNTP, 200mM KCl, 20mM tetramethyl ammonium chloride, final concentration of 500nM of the primer in system,
Probe final concentration of 250nM in system.It is put into fluorescence quantitative PCR instrument, sets response procedures, start to detect, react item
Part: 50 DEG C, 10-15min；95 DEG C, 3-5min；[95 DEG C, 5-15s；60 DEG C, 45-55s] 40 circulations.Detection probe channel and
Grouping situation is shown in Table 2.
2 detection probe channel of table and grouping
(4) result
After the reaction was completed, testing result is analyzed according to amplification curve diagram and Ct value.The fluorescence curve in the channel FAM
In " S " type curve and CT≤37.3, it is judged as positive；Without typical " S " type amplification or CT > 37.3, and interior target CT≤37.3, sentence
Break as feminine gender.Fluorescence curve is in " S " type curve and CT≤37.8 in the channel CY5, is judged as positive；Without typical " S " type amplification
Or CT > 37.8, and interior target CT≤37.8, it is judged as negative.In the channel JOE fluorescence curve in " S " type curve and CT≤
38.2, it is judged as positive；Without typical " S " type amplification or CT > 38.2, and interior target CT≤38.2, it is judged as negative.It is logical in ROX
Fluorescence curve is in " S " type curve and CT≤38.4 in road, is judged as positive；Without typical " S " type amplification or CT > 38.4, and internal standard
CT≤38.4, be judged as negative.
Embodiment 2
The specificity experiments of primed probe group:
Cross-over experiment is done using each Respirovirus plasmid standard to verify its specificity.
For first group, 96 orifice plates (are only added first in each pipe with corresponding primed probe is added in the hole of a line
4 kinds of primed probes of group, are taken turns with this and are pushed away, and 1-5 row respectively corresponds the primed probe of each group of addition, totally 5 row), same row hole
Middle positive plasmid (i.e. containing first group of 4 kinds of viral plasmid) template that identical virus is added, guarantees each primed probe and 16
Any one in kind virus has independent touch opportunity, and cross-over experiment in this way counts each CT value, the verifying present invention
The specificity of method.Separately add other 5 kinds of pathogen: coronavirus (HKU1), enterovirus E71 type (E71), enterovirus
CA16 type (CA16), mycoplasma pneumoniae (MP) and chlamydia pneumoniae (CP), by carrying out cross-over experiment, verifying with the method for the present invention
The specificity of the method for the present invention.
Multiple fluorescence PCR reaction system is 20uL, specifically: each group of pathogen hybrid dna sample 2uL, 2 ×
PCRbuffer 10uL, mixed enzyme solution 0.5uL, primed probe mixed liquor 2uL, DEPC water is mended to 20uL.It is put into quantitative fluorescent PCR
In instrument, response procedures are set, start to detect, reaction condition: 50 DEG C, 10-15min；95 DEG C, 3-5min；[95 DEG C, 5-15s；
60 DEG C, 45-55s] 40 circulations.It the results are shown in Table 3, the primed probe only corresponding disease in every group it can be seen from 3 result of table
Substance has amplification, shows that each primed probe has good specificity.
The specificity experiments result of 3 the method for the present invention of table
Embodiment 3
Sensitivity experiment
Homemade 16 kinds of Respirovirus detection positive plasmid sample is carried out ten times to dilute, 102-105Copies/ul makes
Sample after above-mentioned dilution is expanded respectively with kit provided by the invention, 16 kinds of viral diagnosis sensitivity results are as schemed
Shown in 1- Figure 16, wherein the R of the standard curve of 16 kinds of Respirovirus testing results in Fig. 1-Figure 162Value is respectively 0.999
(RSVA)、0.999(RSVB)、0.995(FluA)、0.998(FluB)、0.999(HBOV)、0.999(OC43)、0.998
(229E)、0.922(NL63)、0.998(HPIV1)、0.995(HPIV2)、0.999(HPIV3)、0.998(HPIV4)、0.999
(MPV)、0.985(HEV)、0.972(ADV)、0.998(HRV)。
Embodiment 4
Accuracy experiment
Each 10,16 kinds of viral samples are randomly choosed, is detected with kit of the invention, while sample being sequenced
Resulting sequence is compared through Blast, and this patent kit test result and sequencing result coincidence rate are 100%.
Embodiment 5
Repeated experiment
By 102-105The sample of copies/ul does 3 holes in parallel, calculates 3 pipe sample CTThe coefficient of variation between value.As a result see
The following table 4, the coefficient of variation is 4% hereinafter, illustrate that kit of the present invention has good repeatability.
The kit of the present invention of table 4 detects the repeated result of 16 kinds of viruses
The detection of 6 actual clinical sample of embodiment
From Langfang City in the Nasopharyngeal swabs sample of acquisition, (picker is certainly for district hospital for the used clinical sample of the present embodiment
The principle of hope), totally 537.Extract viral nucleic acid.Carry out multiple fluorescence PCR detection using kit of the invention, by with it is interior
Reference substance and positive reference substance are compared, and are obtained result (as shown in table 5 below), the positive rate detected using kit of the present invention
It is 48.4%, it is consistent with traditional Virus culture method testing result, and it is much better than Virus culture method in detection time.
Testing result of the kit of the present invention of table 5 to clinical sample
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily
Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and legend shown and described herein.
<110>Langfang Nuo Dao Zhong Ke Laboratory of medical test Co., Ltd
<120>a kind of kit and its application for detecting Respirovirus
<160>48
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cacactaaca actgcaatca tacaa 25
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<210>5
<211>22
<212>DNA
<213>artificial sequence
<220>
<400>5
ctaaagacaa gaccaatcyt gt 22
<210>6
<211>25
<212>DNA
<213>artificial sequence
<220>
<400>6
gacaaagcgt ctacgctgca gtcct 25
<210>7
<211>20
<212>DNA
<213>artificial sequence
<220>
<400>7
cagcattaaa atgcaagggt 20
<210>8
<211>20
<212>DNA
<213>artificial sequence
<220>
<400>8
tccacctact tcgttccccc 20
<210>9
<211>20
<212>DNA
<213>
<220>
<400>9
cagtggggag agaggctcgg 20
<210>10
<211>26
<212>DNA
<213>artificial sequence
<220>
<400>10
tgtcccgtag gagaacccct actgag 26
<210>11
<211>27
<212>DNA
<213>artificial sequence
<220>
<400>11
actccctcag ggttactata ttgaagg 27
<210>12
<211>20
<212>DNA
<213>artificial sequence
<220>
<400>12
acgcgatcct gcactagagg 20
<210>13
<211>18
<212>DNA
<213>artificial sequence
<220>
<400>13
ctctttatag ccctttgc 18
<210>14
<211>18
<212>DNA
<213>artificial sequence
<220>
<400>14
gttctgaaac gtttttga 18
<210>15
<211>18
<212>DNA
<213>artificial sequence
<220>
<400>15
aacagtctcg cactcgtt 18
<210>16
<211>18
<212>DNA
<213>artificial sequence
<220>
<400>16
ttatcaaaac ctaagttc 18
<210>17
<211>20
<212>DNA
<213>
<220>
<400>17
accattgcta ttcaccatgc 20
<210>18
<211>25
<212>DNA
<213>artificial sequence
<220>
<400>18
tgagcaacaa ccccaatatc tcatt 25
<210>19
<211>22
<212>DNA
<213>artificial sequence
<220>
<400>19
tgcagttgga agcgggatct at 22
<210>20
<211>23
<212>DNA
<213>artificial sequence
<220>
<400>20
cacaggttat gttgggatgt ttt 23
<210>21
<211>22
<212>DNA
<213>artificial sequence
<220>
<400>21
gaaaacactg accccagaac aa 22
<210>22
<211>19
<212>DNA
<213>artificial sequence
<220>
<400>22
actgccgctg taatttgtg 19
<210>23
<211>20
<212>DNA
<213>artificial sequence
<220>
<400>23
cttatgattg ctgccagagc 20
<210>24
<211>22
<212>DNA
<213>artificial sequence
<220>
<400>24
tcttggatta atggttaatg ta 22
<210>25
<211>17
<212>DNA
<213>
<220>
<400>25
tagacattat gccacca 17
<210>26
<211>21
<212>DNA
<213>artificial sequence
<220>
<400>26
gggcatgagc attttcccta a 21
<210>27
<211>17
<212>DNA
<213>artificial sequence
<220>
<400>27
gtcctccggc ccctgaa 17
<210>28
<211>19
<212>DNA
<213>artificial sequence
<220>
<400>28
taagcagcca aacaataag 19
<210>29
<211>16
<212>DNA
<213>artificial sequence
<220>
<400>29
cagtgggcgt acatgc 16
<210>30
<211>17
<212>DNA
<213>artificial sequence
<220>
<400>30
tcacatcgtg ggtcggg 17
<210>31
<211>19
<212>DNA
<213>artificial sequence
<220>
<400>31
agccatacat tggacaggg 19
<210>32
<211>20
<212>DNA
<213>artificial sequence
<220>
<400>32
ggtcccatcc cacaattact 20
<210>33
<211>30
<212>DNA
<213>
<220>
<400>33
atgcaacgaa ccagatcaag aacacaaccc 30
<210>34
<211>26
<212>DNA
<213>artificial sequence
<220>
<400>34
ccagaaaggg ttagcccatc caaaca 26
<210>35
<211>24
<212>DNA
<213>artificial sequence
<220>
<400>35
gctcactggg cacggtgagc gtga 24
<210>36
<211>21
<212>DNA
<213>artificial sequence
<220>
<400>36
agttttgggc tccgatgacc a 21
<210>37
<211>29
<212>DNA
<213>artificial sequence
<220>
<400>37
tcatatcatc aggaacaccc aatcagcca 29
<210>38
<211>26
<212>DNA
<213>artificial sequence
<220>
<400>38
atgtgcgcga agtagatctg gaatta 26
<210>39
<211>22
<212>DNA
<213>artificial sequence
<220>
<400>39
tagcctataa gcttattttt gt 22
<210>40
<211>18
<212>DNA
<213>artificial sequence
<220>
<400>40
tcttcagatc ttgttgct 18
<210>41
<211>26
<212>DNA
<213>
<220>
<400>41
tttctggaga tgtcccgtag gagaac 26
<210>42
<211>27
<212>DNA
<213>artificial sequence
<220>
<400>42
tggtctcata agtgggactc cttccta 27
<210>43
<211>27
<212>DNA
<213>artificial sequence
<220>
<400>43
ggtaattgga actattgctc tgggagt 27
<210>44
<211>25
<212>DNA
<213>artificial sequence
<220>
<400>44
aggatcattg aagttgatgc aatag 25
<210>45
<211>23
<212>DNA
<213>artificial sequence
<220>
<400>45
agataacttc tgatcaacat atc 23
<210>46
<211>23
<212>DNA
<213>artificial sequence
<220>
<400>46
gtcggttccg ctccagactt tcg 23
<210>47
<211>15
<212>DNA
<213>artificial sequence
<220>
<400>47
tacctgagcc cgggt 15
<210>48
<211>18
<212>DNA
<213>artificial sequence
<220>
<400>48
tcatcttgag tcctccgg 18
Claims (7)
1. a kind of for detecting the primer combination of probe of Respirovirus, which is characterized in that the primer combination of probe includes:
1) such as polynucleotide sequence primer pair 1 as shown in SEQ ID NO.1 and SEQ ID NO.2 and by polynucleotide sequence
Probe 1 shown in SEQ ID NO.33；
2) such as polynucleotide sequence primer pair 2 as shown in SEQ ID NO.3 and SEQ ID NO.4 and by polynucleotide sequence
Probe 2 shown in SEQ ID NO.34；
3) as polynucleotide sequence 3 sequence of primer pair as shown in SEQ ID NO.5 and SEQ ID NO.6 and by polynucleotides sequence
Arrange the probe 3 as shown in SEQ ID NO.35；
4) as polynucleotide sequence 4 sequence of primer pair as shown in SEQ ID NO.7 and SEQ ID NO.8 and by polynucleotides sequence
Arrange the probe 4 as shown in SEQ ID NO.36；
5) as polynucleotide sequence 5 sequence of primer pair as shown in SEQ ID NO.9 and SEQ ID NO.10 and by polynucleotides
Sequence probe 5 as shown in SEQ ID NO.37；
6) as polynucleotide sequence 6 sequence of primer pair as shown in SEQ ID NO.11 and SEQ ID NO.12 and by polynucleotides
Sequence probe 6 as shown in SEQ ID NO.38；
7) as polynucleotide sequence 7 sequence of primer pair as shown in SEQ ID NO.13 and SEQ ID NO.14 and by polynucleotides
Sequence probe 7 as shown in SEQ ID NO.39；
8) as polynucleotide sequence 8 sequence of primer pair as shown in SEQ ID NO.15 and SEQ ID NO.16 and by polynucleotides
Sequence probe 8 as shown in SEQ ID NO.40；
9) as polynucleotide sequence 9 sequence of primer pair as shown in SEQ ID NO.17 and SEQ ID NO.18 and by polynucleotides
Sequence probe 9 as shown in SEQ ID NO.41；
10) as polynucleotide sequence 10 sequence of primer pair as shown in SEQ ID NO.19 and SEQ ID NO.20 and by multicore glycosides
Acid sequence probe 10 as shown in SEQ ID NO.42；
11) as polynucleotide sequence 11 sequence of primer pair as shown in SEQ ID NO.21 and SEQ ID NO.22 and by multicore glycosides
Acid sequence probe 11 as shown in SEQ ID NO.43；
12) as polynucleotide sequence 12 sequence of primer pair as shown in SEQ ID NO.23 and SEQ ID NO.24 and by multicore glycosides
Acid sequence probe 12 as shown in SEQ ID NO.44；
13) as polynucleotide sequence 13 sequence of primer pair as shown in SEQ ID NO.25 and SEQ ID NO.26 and by multicore glycosides
Acid sequence probe 13 as shown in SEQ ID NO.45；
14) as polynucleotide sequence 14 sequence of primer pair as shown in SEQ ID NO.27 and SEQ ID NO.28 and by multicore glycosides
Acid sequence probe 14 as shown in SEQ ID NO.46；
15) as polynucleotide sequence 15 sequence of primer pair as shown in SEQ ID NO.29 and SEQ ID NO.30 and by multicore glycosides
Acid sequence probe 15 as shown in SEQ ID NO.47；
16) as polynucleotide sequence 16 sequence of primer pair as shown in SEQ ID NO.31 and SEQ ID NO.32 and by multicore glycosides
Acid sequence probe 16 as shown in SEQ ID NO.48.
2. as described in claim 1 for detecting the primer combination of probe of Respirovirus, which is characterized in that the probe
5 ' ends are marked with any one of fluorescent reporter group FAM, JOE, ROX or Cy5；3 ' ends of the probe are marked with fluorescent quenching
Group BHQ1 or BHQ2.
3. the kit for detecting Respirovirus, which is characterized in that including the described in any item use of such as claims 1 or 2
In the primer combination of probe of detection Respirovirus.
4. as claimed in claim 3 for detecting the kit of Respirovirus, which is characterized in that further include negative control,
Positive control, internal reference, reaction amplification liquid, mixed enzyme solution and DEPC water；Wherein, mixed enzyme solution includes Taq DNA enzymatic, reverse transcription
Enzyme and RNase inhibitor；Negative control is DEPC water；Positive control is the matter of the extension increasing sequence comprising 16 kinds of Respirovirus
Grain, internal reference are source of people beta-globin.
5. as claimed in claim 4 for detecting the kit of Respirovirus, which is characterized in that the Respirovirus is
Respiratory Syncytial Virus(RSV) A, Respiratory Syncytial Virus(RSV) B, influenza virus A, influenza virus B, Coronavirus OC43,
Coronavirus 229E, coronavirus N L63, human metapneumovirus, bocavirus, enterovirus, parainfluenza virus 1, parainfluenza virus
2, parainfluenza virus 3, parainfluenza virus 4, rhinovirus and adenovirus.
6. the kit as claimed in claim 3 for detecting Respirovirus is in detecting or assisting detection Respirovirus
Non-diagnostic purpose application.
7. application as claimed in claim 6, which is characterized in that the kit is detecting or assisting detection Respirovirus
Reaction system is 20uL, specifically: sample 2uL to be detected, 2 × PCR buffer 10uL, mixed enzyme solution 0.5uL, primer are visited
Needle combines liquid 2uL, mends DEPC water to 20uL；
Wherein, final concentration of 100-1000nM of the primer pair in amplification system；Final concentration of 50- of the probe in amplification system
500nM。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910677694.5A CN110358865A (en) | 2019-07-25 | 2019-07-25 | For detecting kit and its application of Respirovirus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910677694.5A CN110358865A (en) | 2019-07-25 | 2019-07-25 | For detecting kit and its application of Respirovirus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110358865A true CN110358865A (en) | 2019-10-22 |
Family
ID=68221964
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910677694.5A Pending CN110358865A (en) | 2019-07-25 | 2019-07-25 | For detecting kit and its application of Respirovirus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110358865A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111808989A (en) * | 2020-06-18 | 2020-10-23 | 重庆浦洛通基因医学研究院有限公司 | Coronavirus/influenza virus/rhinovirus nucleic acid combined detection kit and use method thereof |
| CN114540544A (en) * | 2021-12-24 | 2022-05-27 | 廊坊诺道中科医学检验实验室有限公司 | Primer-probe combination for detecting respiratory viruses, kit and application thereof |
| CN116287434A (en) * | 2022-10-21 | 2023-06-23 | 深圳康美生物科技股份有限公司 | Respiratory tract syndrome pathogen nucleic acid detection kit |
| CN116676429A (en) * | 2023-07-27 | 2023-09-01 | 广东省林业科学研究院 | LAMP primer group and method for detecting pangolin respiratory syncytial virus A and B |
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| CN107058622A (en) * | 2017-03-30 | 2017-08-18 | 德必碁生物科技（厦门）有限公司 | A kind of kit of multiple fluorescence PCR method joint-detection respiratory pathogen |
| CN107475446A (en) * | 2017-08-24 | 2017-12-15 | 复旦大学附属儿科医院 | Multi-PCR detection method and its probe groups and kit a kind of while that detect various respiratory road virus |
| CN109207639A (en) * | 2018-10-22 | 2019-01-15 | 南通国际旅行卫生保健门诊部 | Respiratory pathogen rapid fluorescence PCR detection kit and its primer combination of probe |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058622A (en) * | 2017-03-30 | 2017-08-18 | 德必碁生物科技（厦门）有限公司 | A kind of kit of multiple fluorescence PCR method joint-detection respiratory pathogen |
| CN107475446A (en) * | 2017-08-24 | 2017-12-15 | 复旦大学附属儿科医院 | Multi-PCR detection method and its probe groups and kit a kind of while that detect various respiratory road virus |
| CN109207639A (en) * | 2018-10-22 | 2019-01-15 | 南通国际旅行卫生保健门诊部 | Respiratory pathogen rapid fluorescence PCR detection kit and its primer combination of probe |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111808989A (en) * | 2020-06-18 | 2020-10-23 | 重庆浦洛通基因医学研究院有限公司 | Coronavirus/influenza virus/rhinovirus nucleic acid combined detection kit and use method thereof |
| CN114540544A (en) * | 2021-12-24 | 2022-05-27 | 廊坊诺道中科医学检验实验室有限公司 | Primer-probe combination for detecting respiratory viruses, kit and application thereof |
| CN116287434A (en) * | 2022-10-21 | 2023-06-23 | 深圳康美生物科技股份有限公司 | Respiratory tract syndrome pathogen nucleic acid detection kit |
| CN116676429A (en) * | 2023-07-27 | 2023-09-01 | 广东省林业科学研究院 | LAMP primer group and method for detecting pangolin respiratory syncytial virus A and B |
| CN116676429B (en) * | 2023-07-27 | 2023-11-14 | 广东省林业科学研究院 | LAMP primer group for detecting pangolin respiratory syncytial virus type B and application thereof |
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