CN108676913A - A kind of human parainfluenza viruses' nucleic acid is hands-free to take gene parting detecting reagent - Google Patents
A kind of human parainfluenza viruses' nucleic acid is hands-free to take gene parting detecting reagent Download PDFInfo
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- CN108676913A CN108676913A CN201810135541.3A CN201810135541A CN108676913A CN 108676913 A CN108676913 A CN 108676913A CN 201810135541 A CN201810135541 A CN 201810135541A CN 108676913 A CN108676913 A CN 108676913A
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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
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- C12Q1/701—Specific hybridization probes
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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]
Abstract
It takes the invention discloses a kind of human parainfluenza viruses' nucleic acid is hands-free, rapid gene parting detecting reagent, including PCR amplification reagent and contrast agents, the PCR amplification reagent is made of PCR reaction buffers A, enzyme system B and HPIV1/2/3/4 primed probe mixed liquor, and the contrast agents are mainly made of negative control and positive control.The present invention has many advantages, such as that sample directly expands, Multiple detection speed is fast, high sensitivity, specificity are good.
Description
Technical field
The present invention relates to biotechnology and medical domain, specifically a kind of human parainfluenza viruses' nucleic acid parting detection examinations
Agent box.
Background technology
Human parainfluenza viruses (human parainfluenza virus, HPIV) belong to Paramyxoviridae, are that negativity is single-stranded
RNA virus is common Community Acquired Respiratory Tract Infection cause of disease, because its many characteristic is different from influenza virus, and again successively
Other isolated strains, therefore be named as parainfluenza virus, according to science of heredity and Serological Characterization, HPIV can be divided into 1 type, and 2
Type, 3 types and 4 types.
Parainfluenza virus infection whole year can occur (especially the 3rd type), when autumn, croup or laryngotracheobronchitis
(the 1st, 2 type) can be popular in children, then while infecting it is usually slight upper respiratory disease it is more common.However, same type
Parainfluenza virus, especially the 1st type and the 3rd type can cause repeatedly to infect.1st and the 3rd type parainfluenza virus infection Chang Liuhang hairs
It is born in nursery, the children such as primary school place, the 3rd type is endemic conditions, and infectiousness is strong, and the four seasons can occur, most children 1 years old
Interior to infect, the epidemic disease caused by haemadsorption virus 2 or 2 types has can often occur every year and alternating is prevailing
Tendency.Usual haemadsorption virus 1 incubation period is 24~48 hours, and 1 type is 4~5 days.Disease early stage have moderate sore-throat and
Dry cough, many case hoarsenesses and croup symptom protrude.
Human parainfluenza viruses are present in respiratory secretions, are passed through respiratory tract with droplet by being in direct contact for person to person
It broadcasts.For adult, HPIV mainly invades the textura epidermoidea of respiratory mucosa, is proliferated in epithelial cell, and caused lesion is lighter,
Normally behave as the infection of the upper respiratory tract (URI).Infant less than 5 years old, it is thin that virus often invades trachea-bronchial epithelial cell mucosal epithelium
Born of the same parents cause cell degeneration, downright bad hyperplasia and mucosal erosion.When invading alveolar epithelium and interstitial cell, then cause interstitial pneumonia
Or show as acute obstructive laryngotracheobronchitis and pneumonia.HPIV infection can generate local antibody and serum antibody.People's pair
Up to the present the immunity degradation of influenza infection descendant not yet finds effective prevent and specific treatment method.
Infection symptoms caused by parainfluenza virus are similar with influenza virus, only rely on the detection method of clinical symptoms and routine
The determination for carrying out virus causing disease is unreliable.Therefore Rapid＆Early diagnosis respiratory virus infection can instruct clinic to control in time
It treats, foundation is provided, and is of great significance in terms of avoiding abuse of antibiotics to reasonably select antiviral, anti-bacterial drug, mesh
Before, there are many method for detecting virus infection, and respiratory virus infection viral diagnosis method includes mainly Virus culture, immune glimmering
Light technology, serological test, conventional molecular biological method etc.,
Currently, needing progress nucleic acid extraction, substance detection, sensitivity not high using existing detection kit generally existing
And the problems such as time-consuming, it needs effectively to be improved.
Invention content
The purpose of the present invention is to solve samples in the prior art directly to expand, time-consuming for substance detection, detection effect
The lower defect of rate, provides that a kind of human parainfluenza viruses' nucleic acid is hands-free to be taken, and rapid gene parting detecting reagent is above-mentioned to solve
Problem.
To achieve the goals above, the technical solution of invention is as follows：The invention discloses a kind of human parainfluenza viruses' nucleic acid
Parting detecting reagent, including PCR amplification reagent and contrast agents, the PCR amplification reagent is by PCR reaction buffers A, enzyme
It is that B and HPIV1/2/3/4 primed probe mixed liquors are constituted, the contrast agents are mainly by negative control and positive controls
At.
Preferably, the ingredient of the PCR reaction buffers A is 50mM Tris-HCl, 75mM KCl, 3.5mM
MgCl2,10mM DTT, 3% formamide, 0.3mg/ml BSA, the enzyme system B are mainly gathered by HotStart Hi Taq DNA
Synthase, Super-M-MLV reverse transcriptase, RNase inhibitor are constituted, and the HPIV1/2/3/4 primed probe mixed liquors are main
It is composed of the probe and primer that mark the human parainfluenza viruses 1/2/3/4 of different fluorescent reporter groups.
Preferably, the HPIV1 types, the particular sequence of HPIV2 types, HPIV3 types, the probe of HPIV4 types and primer
It is as follows with serial number：
1 type sense primers of HPIV：
AGAACTAGGTGTGACAGACACA SEQ ID NO.1
1 type upstream and downstream primers of HPIV：
CCCTATCAGCRGTGTCCT SEQ ID NO.2
Probe in 1 types of HPIV：
5 '-fluorescence report group-CCACTTCTATRGCTCCACCACCTGTG- fluorescent quenchings group -3 ' SEQ ID NO.3
2 type of human parainfluenza viruses
2 type sense primers of HPIV：
GGCAATGGGCCACAATCAATC SEQ ID NO.4
2 type downstream primers of HPIV：
GCGACCACCATATACAGGAAAT SEQ ID NO.5
2 type probes of HPIV：
5 '-fluorescence report group-CCTAGATGATAGATCCCGCTTCCAACTG- fluorescent quenchings group -3 ' SEQ ID
NO.6
3 type sense primers of HPIV：
CATTCATCTGTATCCTCAGAG SEQ ID NO.7
3 type downstream primers of HPIV：
CCACTCCCATTGCATAACTCC SEQ ID NO.8
3 type probes of HPIV：
5 '-fluorescence report group-AGTTGCCTGGTGCGAACTCACCATG- fluorescent quenchings group -3 ' SEQ ID NO.9
4 type of human parainfluenza viruses
HPIV4 type sense primers：
CTGAAGAGAGAMGRCTGGCYAAG SEQ ID NO.10
HPIV4 type downstream primers：
AATTTTCCTKGCATCTGTTTG SEQ ID NO.11
HPIV4 type probes：
5 '-fluorescence report group-TCYAACATCCTRCCCTGCTGCTTGT- fluorescent quenchings group -3 ' SEQ ID
NO.12。
Preferably, the fluorescent reporter group is selected from FAM, HEX, ROX, CY3, CY5 fluorescent reporter group, it is described
Quenching group is selected from BHQ1, BHQ2, TAMRA fluorescent quenching group.
Preferably, in the HPIV1/2/3/4 primed probe mixed liquors, the 1 type primed probe ratios of HPIV
For：SEQ ID NO.1：SEQ ID NO.2：SEQ ID NO.3 are 400nM：400nM：200nM；The 2 type primers of HPIV
Probe ratio is：SEQ ID NO.4：SEQ ID NO.5：SEQ ID NO.6 are 600nM：600nM：300nM；The HPIV
3 type primed probe ratios are：SEQ ID NO.7：SEQ ID NO.8：SEQ ID NO.9 are 300nM：300nM：100nM；It is described
4 type primed probe ratios of HPIV be：SEQ ID NO.10：SEQ ID NO.11：SEQ ID NO.12 are 300nM：300nM：
150nM。
Preferably, the positive reference substance is to contain 1 type target gene fragments of HIPV's shown in SEQ ID NO.13
Plasmid, the plasmid containing 2 type target gene fragments of HIPV shown in SEQ ID NO.14 or plasmid made of being reconstructed through genetic engineering
Bacterium, the plasmid containing 3 type target gene fragments of HIPV shown in SEQ ID NO.15 or plasmid made of being reconstructed through genetic engineering
Bacterium, the plasmid containing 4 type target gene fragments of HIPV shown in SEQ ID NO.16 or plasmid bacterial made of being reconstructed through genetic engineering
In one or more arbitrary proportions combination..
Preferably, the 1 type target gene fragment sequences of HPIV are：
GCAGTGCTCTGGAGGAAGAACTAGGTGTGACAGACACAGCAAAAGAGAGACTAAGACACCATCTAACAA
ACCTTTCAGGAGGGGATGGTGCCTACCATAAACCCACAGGTGGTGGAGCTATAGAAGTGGCAATTGATCATACAGAC
ATAACATTTGGAGCAGAGGACACCGCTGATAGGGACAACAAAAACTGGGCAAATGACAGCAACGAAAGATGGATGAA
TCACTCTACCAACAACCACACAAT
SEQ ID NO.13
The 2 type target gene fragment sequences of HPIV are：
ACAACAGGGCAATGGGCCACAATCAATCCTGCAGTTGGAAGCGGGATCTATCATCTAGGCTTTATTTTA
TTTCCTGTATATGGTGGTCTCATAAATGGGACTCCTTCCTACAACGAGCAGTCCTCACGCTACTTTATCCCAACACA
TCCCAACATAACCTGTGCCGGTAACTCCAGTGTACAGGCTGCAGCAGCACGGAGTTCCTAT
SEQ ID NO.14
The 3 type target gene fragment sequences of HPIV are：
GCGCTCCATTCATCTGTATCCTCAGAGATCCCATACATGGTGAGTTTGCACCAGGCAACTATCCTGCCA
TATGGAGTTATGCAATGGGAGTGGCAGTTGTACA
SEQ ID NO.15
The 4 type target gene fragment sequences of HPIV are：
ATCAACCCACTGGATCTGAAGAGAGACGACTGGCTAAGTACAAGCAGCAGGGCAGGATGTTAGAGAAAT
ATCAACTGCAAACAGATGCCAGGAAAATTATCCAATTAGTAATAAGAGAAAGTATGGTTATAAGGCAATTTCTTGTA
CAGGAGA
SEQ ID NO.16
It is a kind of that human parainfluenza viruses' nucleic acid parting is carried out using above-mentioned human parainfluenza viruses' nucleic acid parting detecting reagent
Detection, detection method are as follows：
(1) sample prepares
Nucleic acid extraction is carried out, the viral RNA of throat swab sample and serum sample can directly be expanded；
(2) preparation of PCR reaction systems
Prepare PCR reaction systems, wherein 19 μ l of HPIV PCR reaction buffers A, 2 μ l of enzyme system B, primed probe mixed liquor
PCR reaction solution is dispensed by 23 μ l/ pipes into PCR reaction tubes, the reaction tube equipped with PCR reaction solution is moved to sample process by 2 μ l
Area；
(3) it is loaded
Each 2 μ l of processed sample, negative control, positive control supernatant are taken to be added separately to respectively with the suction nozzle with filter core
In PCR reaction tubes equipped with reaction system, lid upper tube cap moves to augmentation detection area after centrifuging the several seconds；
(4) it expands
PCR reaction tubes are put into fluorescent PCR amplification instrument and carry out augmentation detection.
Preferably, in the step (4), loop parameter setting is as follows：(AB：ABI 7500FAST, Anlong：Pro
Eco48, SLAN-96)：
The present invention has the following advantages compared with prior art：
1, hands-free to take, for the viral RNA of throat swab sample and serum sample be not necessarily to nucleic acid extraction, can directly to sample into
Row amplification；
2, multiple real time fluorescence quantifying PCR expands, and a pipe PCR can detect the human parainfluenza viruses of 4 kinds of types, convenient, fast
Prompt, saving；
3, accuracy is good, carries out parting detection using taqman sonde methods, ensures the reliability of testing result；
4, precision is high, and kit repeats detection 10 times, intermediate value cv to intermediate value and low value precision standard items<1%, low value
cv<2%.
Description of the drawings
Fig. 1 is human parainfluenza viruses' nucleic acid parting detecting reagent of the present invention to HPIV1 type positive clinical samples
Directly amplification and nucleic acid extraction amplification；
Fig. 2 is human parainfluenza viruses' nucleic acid parting detecting reagent of the present invention and company A kit to HPIV1 types
The direct amplification of positive clinical sample；
Fig. 3 is that human parainfluenza viruses' nucleic acid parting detecting reagent of the present invention detects 1 type enterprises of HPIV self-control essence
Density criterion product precision result figure；
Fig. 4 is that human parainfluenza viruses' nucleic acid parting detecting reagent of the present invention detects 2 type enterprises of HPIV self-control essence
Density criterion product precision result figure；
Fig. 5 is that human parainfluenza viruses' nucleic acid parting detecting reagent of the present invention detects 3 type enterprises of HPIV self-control essence
Density criterion product precision result figure；
Fig. 6 is that human parainfluenza viruses' nucleic acid parting detecting reagent of the present invention detects 4 type enterprises of HPIV self-control essence
Density criterion product precision result figure；
Fig. 7 is that human parainfluenza viruses' nucleic acid parting detecting reagent of the present invention detects specific outcome figure.
Specific implementation mode
To make the structure feature to invention and the effect of being reached has a better understanding and awareness, to preferably real
Example and attached drawing cooperation detailed description are applied, is described as follows：
The invention discloses a kind of human parainfluenza viruses' nucleic acid parting detecting reagents, including PCR amplification reagent and control
Reagent, the PCR amplification reagent are made of PCR reaction buffers A, enzyme system B and HPIV1/2/3/4 primed probe mixed liquor,
The contrast agents are mainly made of negative control and positive control.
Preferably, the ingredient of the PCR reaction buffers A is 50mM Tris-HCl, 75mM KCl, 3.5mM
MgCl2,10mM DTT, 3% formamide, 0.3mg/ml BSA, the enzyme system B are mainly gathered by HotStart Hi Taq DNA
Synthase, Super-M-MLV reverse transcriptase, RNase inhibitor are constituted, and the HPIV1/2/3/4 primed probe mixed liquors are main
It is composed of the probe and primer that mark the human parainfluenza viruses 1/2/3/4 of different fluorescent reporter groups.
Wherein, the destruction cell membrane of formamide main function discharges nucleic acid, makes albuminous degeneration；The main function of BSA is dimension
Hold the stability and enzymatic activity of taq enzymes.
Preferably, the HPIV1 types, the particular sequence of HPIV2 types, HPIV3 types, the probe of HPIV4 types and primer
It is as follows with serial number：
1 type sense primers of HPIV：
AGAACTAGGTGTGACAGACACA SEQ ID NO.1
1 type upstream and downstream primers of HPIV：
CCCTATCAGCRGTGTCCT SEQ ID NO.2
Probe in 1 types of HPIV：
5 '-fluorescence report group-CCACTTCTATRGCTCCACCACCTGTG- fluorescent quenchings group -3 ' SEQ ID NO.3
2 type of human parainfluenza viruses
2 type sense primers of HPIV：
GGCAATGGGCCACAATCAATC SEQ ID NO.4
2 type downstream primers of HPIV：
GCGACCACCATATACAGGAAAT SEQ ID NO.5
2 type probes of HPIV：
5 '-fluorescence report group-CCTAGATGATAGATCCCGCTTCCAACTG- fluorescent quenchings group -3 ' SEQ ID
NO.6
3 type sense primers of HPIV：
CATTCATCTGTATCCTCAGAG SEQ ID NO.7
3 type downstream primers of HPIV：
CCACTCCCATTGCATAACTCC SEQ ID NO.8
3 type probes of HPIV：
5 '-fluorescence report group-AGTTGCCTGGTGCGAACTCACCATG- fluorescent quenchings group -3 ' SEQ ID NO.9
4 type of human parainfluenza viruses
HPIV4 type sense primers：
CTGAAGAGAGAMGRCTGGCYAAG SEQ ID NO.10
HPIV4 type downstream primers：
AATTTTCCTKGCATCTGTTTG SEQ ID NO.11
HPIV4 type probes：
5 '-fluorescence report group-TCYAACATCCTRCCCTGCTGCTTGT- fluorescent quenchings group -3 ' SEQ ID
NO.12。
Preferably, the fluorescent reporter group is selected from FAM, HEX, ROX, CY3, CY5 fluorescent reporter group, it is described
Quenching group is selected from BHQ1, BHQ2, TAMRA fluorescent quenching group.
Preferably, in the HPIV1/2/3/4 primed probe mixed liquors, the 1 type primed probe ratios of HPIV
For：SEQ ID NO.1：SEQ ID NO.2：SEQ ID NO.3 are 400nM：400nM：200nM；The 2 type primers of HPIV
Probe ratio is：SEQ ID NO.4：SEQ ID NO.5：SEQ ID NO.6 are 600nM：600nM：300nM；The HPIV
3 type primed probe ratios are：SEQ ID NO.7：SEQ ID NO.8：SEQ ID NO.9 are 300nM：300nM：100nM；It is described
4 type primed probe ratios of HPIV be：SEQ ID NO.10：SEQ ID NO.11：SEQ ID NO.12 are 300nM：300nM：
150nM。
Preferably, the positive reference substance is to contain 1 type target gene fragments of HIPV's shown in SEQ ID NO.13
Plasmid, the plasmid containing 2 type target gene fragments of HIPV shown in SEQ ID NO.14 or plasmid made of being reconstructed through genetic engineering
Bacterium, the plasmid containing 3 type target gene fragments of HIPV shown in SEQ ID NO.15 or plasmid made of being reconstructed through genetic engineering
Bacterium, the plasmid containing 4 type target gene fragments of HIPV shown in SEQ ID NO.16 or plasmid bacterial made of being reconstructed through genetic engineering
In one or more arbitrary proportions combination..
Preferably, the 1 type target gene fragment sequences of HPIV are：
GCAGTGCTCTGGAGGAAGAACTAGGTGTGACAGACACAGCAAAAGAGAGACTAAGACACCATCTAACAA
ACCTTTCAGGAGGGGATGGTGCCTACCATAAACCCACAGGTGGTGGAGCTATAGAAGTGGCAATTGATCATACAGAC
ATAACATTTGGAGCAGAGGACACCGCTGATAGGGACAACAAAAACTGGGCAAATGACAGCAACGAAAGATGGATGAA
TCACTCTACCAACAACCACACAAT
SEQ ID NO.13
The 2 type target gene fragment sequences of HPIV are：
ACAACAGGGCAATGGGCCACAATCAATCCTGCAGTTGGAAGCGGGATCTATCATCTAGGCTTTATTTTA
TTTCCTGTATATGGTGGTCTCATAAATGGGACTCCTTCCTACAACGAGCAGTCCTCACGCTACTTTATCCCAACACA
TCCCAACATAACCTGTGCCGGTAACTCCAGTGTACAGGCTGCAGCAGCACGGAGTTCCTAT
SEQ ID NO.14
The 3 type target gene fragment sequences of HPIV are：
GCGCTCCATTCATCTGTATCCTCAGAGATCCCATACATGGTGAGTTTGCACCAGGCAACTATCCTGCCA
TATGGAGTTATGCAATGGGAGTGGCAGTTGTACA
SEQ ID NO.15
The 4 type target gene fragment sequences of HPIV are：
ATCAACCCACTGGATCTGAAGAGAGACGACTGGCTAAGTACAAGCAGCAGGGCAGGATGTTAGAGAAAT
ATCAACTGCAAACAGATGCCAGGAAAATTATCCAATTAGTAATAAGAGAAAGTATGGTTATAAGGCAATTTCTTGTA
CAGGAGA
SEQ ID NO.16
It is a kind of that human parainfluenza viruses' nucleic acid parting is carried out using above-mentioned human parainfluenza viruses' nucleic acid parting detecting reagent
Detection, detection method are as follows：
(3), sample prepares
Nucleic acid extraction is carried out, the viral RNA of throat swab sample and serum sample can directly be expanded；
(4), the preparation of PCR reaction systems
Prepare PCR reaction systems, wherein 19 μ l of HPIV PCR reaction buffers C, 2 μ l of enzyme system B, primed probe mixed liquor
PCR reaction solution is dispensed by 23 μ l/ pipes into PCR reaction tubes, the reaction tube equipped with PCR reaction solution is moved to sample process by 2 μ l
Area；
(3), it is loaded
Each 2 μ l of processed sample, negative control, positive control supernatant are taken to be added separately to respectively with the suction nozzle with filter core
In PCR reaction tubes equipped with reaction system, lid upper tube cap moves to augmentation detection area after centrifuging the several seconds；
(5), it expands
PCR reaction tubes are put into fluorescent PCR amplification instrument and carry out augmentation detection.
Preferably, in the step (4), loop parameter setting is as follows：(AB：ABI 7500FAST, Anlong：Pro
Eco48, SLAN-96)：
Embodiment 1
One, sample preparation is carried out in sample process area
1.1, this kit is not necessarily to nucleic acid extraction, can directly expand inspection to the viral nucleic acid of throat swab sample and serum sample
It surveys, only needing to thaw in throat swab sample before experiment is vortexed, and blood sample, which need to centrifuge, obtains serum；
Two, PCR reagent preparation is carried out in reagent area in preparation
2.1, it presses following composition and prepares PCR reaction solution (n is reaction tube number)：
HPIV PCR reaction buffers 1 are that 19 μ l × n, HPIV1/2/3/4 probe mixed liquors are 2 μ l × n, and enzyme system 3 is 2 μ l
× n wherein ensures that PCR reaction buffers, primed probe mixed liquor fully dissolve before use, enzyme system needs centrifugation with true before use
It protects all enzymes and concentrates on bottom；
2.2, PCR reaction solution is dispensed by 23 μ l/ pipes into PCR reaction tubes, the reaction tube equipped with PCR reaction solution is moved to
Sample process area；
Three, it is loaded in sample process area
Each 2 μ l of processed sample, negative control, positive control supernatant are taken to be added separately to respectively with the suction nozzle with filter core
In PCR reaction tubes equipped with PCR reaction solution, lid upper tube cap moves to augmentation detection area after centrifuging the several seconds；
Four, PCR amplification detection is carried out in augmentation detection area
4.1, PCR reaction tubes are put into fluorescent PCR amplification instrument and carry out augmentation detection；
4.2, loop parameter is set：(ABI 7500, SLAN96P)：
Five, quality control standard
5.1, negative control：As a result it is feminine gender；
5.2, positive control：As a result it is the positive, value≤33 positive control Ct；
5.3, above two need meet simultaneously in one experiment, and otherwise, this experiment is invalid, and experiment should re-start；
Six, the explanation of inspection result
6.1, experiment is both needed to detect negative control, the positive control in kit together with sample to be checked every time, detects
As a result it should meet its performance indicator, otherwise this experiment is invalid；
6.2, experimental result judgment method
(1), negative findings judge：If sample, in the channels FAM, the channels HEX/VIC, the channels ROX and the amplification of the channels Cy5 are bent
Line is UNDET not at S types, Ct values, then result is feminine gender；
(2), positive findings judge：If sample is S-type in the channels FAM amplification curve and value≤38 Ct, result are pair
1 type of influenza virus is positive, if sample is S-type in the channels HEX/VIC amplification curve and value≤38 Ct, result are parainfluenza virus
Malicious 2 types are positive, if sample is S-type in the channels ROX amplification curve and value≤38 Ct, result are that haemadsorption virus 1 is positive,
If sample is S-type in the channels Cy5 amplification curve and value≤38 Ct, result are that parainfluenza virus type 4 is positive；
(3), gray area is tested：If sample is in the channels FAM, the channels HEX/VIC, the channels ROX or the channels Cy5 amplification curve
It is S-type and 38<Ct values<40, then result, which is located at, tests gray area, should be rechecked to sample.
(4), " reinspection " operation is as follows：It is recommended that carrying out concentration to sample, the preservation liquid after sample is vortexed fully is collected
After 4 DEG C of centrifugation 5min of 1ml, 12000rpm, supernatant is carefully removed, the about 200 μ l of layer that keep on file are as sample, and subsequent processing is the same as general
Sample to be tested processing method judges result for the positive if reinspection result amplification curve is S-type, if amplification curve is not S-type,
Then judge result for feminine gender.
Embodiment 2
Respectively use 3 batches human parainfluenza viruses RNA detection kits to positive control, negative control, in, low three
The enterprise of a concentration makes standard items by oneself and carries out 10 repetition detections, and the CV of batch interior imprecision Ct values is ranging from：HPIV-1 is
It is 0.77~1.47%, HPIV-4 is 0.89~1.65% that 0.68~1.42%, HPIV-2, which are 0.72~1.55%, HPIV-3,；
Batch between imprecision Ct values CV ranging from：HPIV-1 is that 1.12~1.19%, HPIV-2 is that 1.04~1.40%, HPIV-3 is
1.03~1.52%, HPIV-4 are 0.95~1.40%；In batch and the CV values of betweenrun precision Ct values are respectively less than 5%, and feminine gender is right
According to being feminine gender, show that this kit precision is good, testing result such as the following table 1：
Table 1
Embodiment 3
Selection has homology with human parainfluenza viruses' nucleic acid sequence, easily causes same or analogous clinical symptoms, sampling
The normal parasitic or easily concurrent other pathogens in position, such as influenza A virus, influenza B virus, influenza virus C, nose
Virus, Respiratory Syncytial Virus(RSV), adenovirus, measles virus, rubella virus, mumps virus are sample to be checked, use matter
The qualified human parainfluenza viruses' nucleic acid parting detecting reagent of inspection is detected, and operation is carried out in strict accordance with kit specification,
It is detected on ABI7500 real-time fluorescence quantitative PCR instruments, testing result is feminine gender, shows that kit specificity is good.
The advantages of basic principles and main features and the invention of invention has been shown and described above.The technical staff of the industry
It should be appreciated that invention is not restricted to the described embodiments, what is described in the above embodiment and the description is only the principle of invention,
It invents and will also have various changes and improvements under the premise of not departing from spirit and range, these changes and improvements both fall within requirement
In the range of the invention of protection.The protection domain that invention requires is defined by appended claims and its equivalent.
Claims (10)
1. a kind of human parainfluenza viruses' nucleic acid is hands-free to be taken, rapid gene parting detecting reagent, it is characterised in that：Expand including PCR
Increase reagent and contrast agents, the PCR amplification reagent is by PCR reaction buffers A, enzyme system B and HPIV1/2/3/4 primed probe
Mixed liquor is constituted, and the contrast agents are mainly made of negative control and positive control.
2. a kind of human parainfluenza viruses' nucleic acid parting detecting reagent according to claim 1, it is characterised in that：Described
The ingredient of PCR reaction buffers A be 50mM Tris-HCl, 75mM KCl, 3.5mM MgCl2,10mM DTT, 3% formamide,
0.3mg/ml BSA, the enzyme system B mainly by HotStart Hi Taq archaeal dna polymerases, Super-M-MLV reverse transcriptase,
RNase inhibitor is constituted, and the HPIV1/2/3/4 primed probes mixed liquor is mainly by the people of the different fluorescent reporter groups of label
The probe and primer of parainfluenza virus 1/2/3/4 are formed according to certain concentration combination.
3. 3% formamide is added in PCR reaction buffers A according to claim 2, it is main as ionic surface active agent
To be used for destroying cell membrane, release nucleic acid, make albuminous degeneration；The BSA of the 0.3mg/ml mainly protects the stability of taq enzymes
And activity, the reinforcing agent as PCR；The RNase inhibitor is primarily used to prevent the RNA of RNA enzyme degradation cracking, is convenient for
The formation and amplification of later stage cDNA, other compositions normally expand ingredient as RT-PCR.
4. a kind of human parainfluenza viruses' nucleic acid parting detecting reagent according to claim 1, it is characterised in that：Described
HPIV1 types, HPIV2 types, HPIV3 types, the particular sequence and serial number of the probe of HPIV4 types and primer are as follows：
1 type sense primers of HPIV：
AGAACTAGGTGTGACAGACACA SEQ ID NO.1
1 type upstream and downstream primers of HPIV：
CCCTATCAGCRGTGTCCT SEQ ID NO.2
Probe in 1 types of HPIV：
5 '-fluorescence report group-CCACTTCTATRGCTCCACCACCTGTG- fluorescent quenchings group -3 ' SEQ ID NO.3
2 type of human parainfluenza viruses
2 type sense primers of HPIV：
GGCAATGGGCCACAATCAATC SEQ ID NO.4
2 type downstream primers of HPIV：
GCGACCACCATATACAGGAAAT SEQ ID NO.5
2 type probes of HPIV：
5 '-fluorescence report group-CCTAGATGATAGATCCCGCTTCCAACTG- fluorescent quenchings group -3 ' SEQ ID NO.6
3 type sense primers of HPIV：
CATTCATCTGTATCCTCAGAG SEQ ID NO.7
3 type downstream primers of HPIV：
CCACTCCCATTGCATAACTCC SEQ ID NO.8
3 type probes of HPIV：
5 '-fluorescence report group-AGTTGCCTGGTGCGAACTCACCATG- fluorescent quenchings group -3 ' SEQ ID NO.9
4 type of human parainfluenza viruses
HPIV4 type sense primers：
CTGAAGAGAGAMGRCTGGCYAAG SEQ ID NO.10
HPIV4 type downstream primers：
AATTTTCCTKGCATCTGTTTG SEQ ID NO.11
HPIV4 type probes：
5 '-fluorescence report group-TCYAACATCCTRCCCTGCTGCTTGT- fluorescent quenchings group -3 ' SEQ ID NO.12.
5. a kind of human parainfluenza viruses' nucleic acid parting detecting reagent according to claim 1, it is characterised in that：Described
Fluorescent reporter group be selected from FAM, HEX, ROX, CY3, CY5 fluorescent reporter group, the quenching group be selected from BHQ1, BHQ2,
TAMRA fluorescent quenching groups.
6. a kind of human parainfluenza viruses' nucleic acid parting detecting reagent according to claim 1, it is characterised in that：Described
In HPIV1/2/3/4 primed probe mixed liquors, the 1 type primed probe ratios of HPIV are：SEQ ID NO.1：SEQ ID
NO.2：SEQ ID NO.3 are 400nM：400nM：200nM；The 2 type primed probe ratios of HPIV are：SEQ ID NO.4：
SEQ ID NO.5：SEQ ID NO.6 are 600nM：600nM：300nM；The 3 type primed probe ratios of HPIV are：SEQ
ID NO.7：SEQ ID NO.8：SEQ ID NO.9 are 300nM：300nM：100nM；The 4 type primed probe ratios of HPIV
For：SEQ ID NO.10：SEQ ID NO.11：SEQ ID NO.12 are 300nM：300nM：150nM.
7. a kind of human parainfluenza viruses' nucleic acid parting detecting reagent according to claim 1, it is characterised in that：Described
Positive reference substance is the plasmid containing 1 type target gene fragments of HIPV shown in SEQ ID NO.13, contains SEQ ID NO.14 institutes
Plasmid bacterial made of showing the plasmid of 2 type target gene fragments of HIPV or being reconstructed through genetic engineering contains shown in SEQ ID NO.15
The plasmid of 3 type target gene fragments of HIPV or plasmid bacterial made of being reconstructed through genetic engineering, containing shown in SEQ ID NO.16
One or more arbitrary ratios in the plasmid of 4 type target gene fragments of HIPV or plasmid bacterial made of being reconstructed through genetic engineering
The combination of example.
8. a kind of human parainfluenza viruses' nucleic acid parting detecting reagent according to claim 1, it is characterised in that：Described
1 type target gene fragment sequences of HPIV are：
GCAGTGCTCTGGAGGAAGAACTAGGTGTGACAGACACAGCAAAAGAGAGACTAAGACACCATCTAACAAACCT
TTCAGGAGGGGATGGTGCCTACCATAA
ACCCACAGGTGGTGGAGCTATAGAAGTGGCAATTGATCATACAGACATAACATTTGGAGCAGAGGACACCGCTGATA
GGGACAACAAAAACTGGGCAAATGACAGCAACGAAAGATGGATGAATCACTCTACCAACAACCACACAAT SEQ ID
NO.13
The 2 type target gene fragment sequences of HPIV are：
ACAACAGGGCAATGGGCCACAATCAATCCTGCAGTTGGAAGCGGGATCTATCATCTAGGCTTTATTTTATTTC
CTGTATATGGTGGTCTCATAAATGGGACTCCTTCCTACAACGAGCAGTCCTCACGCTACTTTATCCCAACACATCCC
AACATAACCTGTGCCGGTAACTCCAGTGTACAGGCTGCAGCAGCACGGAGTTCCTAT SEQ ID NO.14
The 3 type target gene fragment sequences of HPIV are：
GCGCTCCATTCATCTGTATCCTCAGAGATCCCATACATGGTGAGTTTGCACCAGGCAACTATCCTGCCATATG
GAGTTATGCAATGGGAGTGGCAGTTGTACA SEQ ID NO.15
The 4 type target gene fragment sequences of HPIV are：
ATCAACCCACTGGATCTGAAGAGAGACGACTGGCTAAGTACAAGCAGCAGGGCAGGATGTTAGAGAAATATCA
ACTGCAAACAGATGCCAGGAAAATTATCCAATTAGTAATAAGAGAAAGTATGGTTATAAGGCAATTTCTTGTACAGG
AGA SEQ ID NO.16。
9. a kind of sick to human parainfluenza using any human parainfluenza viruses' nucleic acid parting detecting reagents of claim 1-7
Malicious nucleic acid parting is detected, it is characterised in that：Detection method is as follows：
(1) sample prepares
Nucleic acid extraction is carried out, the viral RNA of throat swab sample and serum sample can directly be expanded；
(2) preparation of PCR reaction systems
Preparation PCR reaction systems, wherein 19 μ l of HPIV PCR reaction buffers C, 2 μ l of enzyme system B, 2 μ l of primed probe mixed liquor,
PCR reaction solution is dispensed by 23 μ l/ pipes into PCR reaction tubes, the reaction tube equipped with PCR reaction solution is moved into sample process area；
(3) it is loaded
Each 2 μ l of processed sample, negative control, positive control supernatant are taken to be added separately to be equipped with respectively with the suction nozzle with filter core
In the PCR reaction tubes of reaction system, lid upper tube cap moves to augmentation detection area after centrifuging the several seconds；
(4) it expands
PCR reaction tubes are put into fluorescent PCR amplification instrument and carry out augmentation detection.
10. detection method according to claim 8, it is characterised in that：In the step (4), loop parameter is set such as
Under：(AB：ABI 7500FAST, Anlong：Pro Eco48, SLAN-96)：
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108998512A (en) * | 2018-08-14 | 2018-12-14 | 上海芯超生物科技有限公司 | The hands-free kit and detection method for CYP2C19 genetic polymorphism detection taken |
| CN109371127A (en) * | 2018-10-22 | 2019-02-22 | 江苏美因康生物科技有限公司 | The kit and method of a kind of while quick detection UGT1A1*6 type and UGT1A1*28 type gene pleiomorphism |
| CN111172327A (en) * | 2020-03-05 | 2020-05-19 | 杭州丹威生物科技有限公司 | Method and kit for detecting novel coronavirus nucleic acid without taking hands |
| CN111926114A (en) * | 2020-07-15 | 2020-11-13 | 四川大学华西医院 | Multiplex-time PCR (polymerase chain reaction) kit for detecting parainfluenza virus, method and application |
| CN113278738A (en) * | 2021-07-01 | 2021-08-20 | 宁波海尔施基因科技有限公司 | Primer probe composition for parainfluenza virus typing detection and real-time fluorescent quantitative PCR kit |
| CN113322351A (en) * | 2021-05-19 | 2021-08-31 | 北京华诺奥美基因生物科技有限公司 | Real-time fluorescent quantitative PCR (polymerase chain reaction) probe primer group and kit for rapidly and qualitatively typing and detecting four types of human parainfluenza viruses |
| WO2021175298A1 (en) * | 2020-03-05 | 2021-09-10 | 浙江东方基因生物制品股份有限公司 | Novel coronavirus detection reagent and detection method |
| CN113388701A (en) * | 2021-07-19 | 2021-09-14 | 首都儿科研究所 | Primer probe composition and application thereof in preparation of parainfluenza virus typing detection kit |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039538A (en) * | 2015-07-10 | 2015-11-11 | 深圳联合医学科技有限公司 | Reaction liquid and kit for direct fluorescent PCR amplification of blood |
| CN105441589A (en) * | 2015-12-21 | 2016-03-30 | 深圳市生科源技术有限公司 | Human parainfluenza virus I, II, III, and IV-type quadruple-PCR detection kit, and detection method thereof |
| CN105734168A (en) * | 2015-10-28 | 2016-07-06 | 南京美宁康诚生物科技有限公司 | Multiplex PCR detection kit for nucleic acids of twelve respiratory viruses |
-
2018
- 2018-02-09 CN CN201810135541.3A patent/CN108676913A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039538A (en) * | 2015-07-10 | 2015-11-11 | 深圳联合医学科技有限公司 | Reaction liquid and kit for direct fluorescent PCR amplification of blood |
| CN105734168A (en) * | 2015-10-28 | 2016-07-06 | 南京美宁康诚生物科技有限公司 | Multiplex PCR detection kit for nucleic acids of twelve respiratory viruses |
| CN105441589A (en) * | 2015-12-21 | 2016-03-30 | 深圳市生科源技术有限公司 | Human parainfluenza virus I, II, III, and IV-type quadruple-PCR detection kit, and detection method thereof |
Non-Patent Citations (5)
| Title |
|---|
| BECK E.T.ET AL.: "JQ901981.1", 《GENBANK》 * |
| ECHEVARRIA,J.E.ET AL.: "AF039937.1", 《GENBANK》 * |
| GRENINGER A.L. ET AL.: "MF554732.1", 《GENBANK》 * |
| YEA C.ET AL.: "EU627591.1", 《GENBANK》 * |
| 董妍玲、洪华珠: "《基因工程》", 31 January 2013 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108998512A (en) * | 2018-08-14 | 2018-12-14 | 上海芯超生物科技有限公司 | The hands-free kit and detection method for CYP2C19 genetic polymorphism detection taken |
| CN109371127A (en) * | 2018-10-22 | 2019-02-22 | 江苏美因康生物科技有限公司 | The kit and method of a kind of while quick detection UGT1A1*6 type and UGT1A1*28 type gene pleiomorphism |
| CN111172327A (en) * | 2020-03-05 | 2020-05-19 | 杭州丹威生物科技有限公司 | Method and kit for detecting novel coronavirus nucleic acid without taking hands |
| WO2021175298A1 (en) * | 2020-03-05 | 2021-09-10 | 浙江东方基因生物制品股份有限公司 | Novel coronavirus detection reagent and detection method |
| GB2607805A (en) * | 2020-03-05 | 2022-12-14 | Zhejiang Orient Gene Biotech Co Ltd | Novel coronavirus detection reagent and detection method |
| CN111926114A (en) * | 2020-07-15 | 2020-11-13 | 四川大学华西医院 | Multiplex-time PCR (polymerase chain reaction) kit for detecting parainfluenza virus, method and application |
| CN111926114B (en) * | 2020-07-15 | 2023-10-13 | 四川大学华西医院 | Multiplex real-time PCR kit, method and application for detecting parainfluenza virus |
| CN113322351A (en) * | 2021-05-19 | 2021-08-31 | 北京华诺奥美基因生物科技有限公司 | Real-time fluorescent quantitative PCR (polymerase chain reaction) probe primer group and kit for rapidly and qualitatively typing and detecting four types of human parainfluenza viruses |
| CN113278738A (en) * | 2021-07-01 | 2021-08-20 | 宁波海尔施基因科技有限公司 | Primer probe composition for parainfluenza virus typing detection and real-time fluorescent quantitative PCR kit |
| CN113388701A (en) * | 2021-07-19 | 2021-09-14 | 首都儿科研究所 | Primer probe composition and application thereof in preparation of parainfluenza virus typing detection kit |
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