CN105039596A - Kit used for detecting parainfluenza viruses and application thereof - Google Patents
Kit used for detecting parainfluenza viruses and application thereof Download PDFInfo
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- CN105039596A CN105039596A CN201510468275.2A CN201510468275A CN105039596A CN 105039596 A CN105039596 A CN 105039596A CN 201510468275 A CN201510468275 A CN 201510468275A CN 105039596 A CN105039596 A CN 105039596A
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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
- C12Q1/70—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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- 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
Abstract
The invention discloses a kit used for detecting parainfluenza viruses and an application thereof. The kit comprises a primer pair set used for detecting the parainfluenza viruses. The primer pair set is composed of three primer pairs, and the sequences of the primer pair set are the first sequence to the sixth sequence in the sequence table. According to the kit, the high throughput is supported, and parainfluenza virus infections can be rapidly and accurately detected; in the clinical aspect, the detection results of three virus indexes can be obtained in one hour, and the kit is rapider than a real-time fluorescence quantification PCR method commonly adopted at present, and is of great significance in rapid treating and pharmacy guiding assisting. Meanwhile, multi-index detection can be used for regional epidemiology investigating and surveying and regional epidemic surveillance to research the epidemic condition of the parainfluenza virus infections in China.
Description
Technical field
The invention belongs to nucleic acid amplification technologies field, relating to a kind of test kit for detecting parainfluenza virus and application thereof.
Background technology
Human parainfluenza virus (HPIVs) is the one virus often causing children's lower respiratory infection, and it is pathogenic is only second to respiratory syncytial virus (RSV).The same with RSV, human parainfluenza virus can cause the upper respiratory tract infection of recurrent exerbation (as caught a cold and having a sore throat).It also can cause the lower respiratory illness (as pneumonia, bronchitis and bronchiolitis) of serious repeated infection, particularly in the elderly and immune deficiency crowd.
4 types (I type is to IV type) can be divided into from human parainfluenza virus serology.Clinical and the epidemiologic feature that four kinds of hypotypes are had nothing in common with each other.I type and the most typical Clinical symptoms of II type cause children's laryngotracheobronchitis, and I type is the major cause of this children's laryngotracheobronchitis, and II type takes second place.I type and II type all can cause other the upper respiratory tract and lower respiratory illness.Type III often causes pneumonia and bronchiolitis.IV type is difficult to detect, and may be because it seldom causes serious disease.The latent period of human parainfluenza virus is generally at about 1 ~ 7 day.
Rapid&Early diagnosis parainfluenza virus infection can in time guiding clinical treatment for choose reasonable antiviral antibacterial medicine provide according to and prevent in abuse of antibiotics significant.
The method of current detection parainfluenza virus is more, but all Shortcomings, as Electronic Speculum detection method complex and expensive, Viral isolation is consuming time extremely long and false negative is more, after taking cultivation results, usually lose clinical meaning, what euzymelinked immunosorbent assay (ELISA) detected is special viral antibody but once only can detects a kind of virus.And based on the detection of nucleic acids of nucleic acid amplification, due to speed fast (can complete detection in usual 3 hours), highly sensitive, specificity good, becomes gold standard just gradually in field of virus detection.
Rely on the amplification technique (NucleicAcidSequenceBasedAmplification) of nucleotide sequence, i.e. NASBA amplification technique, is mediated by pair of primers, specificity the homogeneous continuously process of single stranded RNA being carried out to constant-temperature amplification in vitro.Under 41 DEG C of constant temperatures, in speed of response, NASBA increases 1h can by template ribonucleic acid amplification about 10
9~ 10
12doubly, common PCR reaction needed 2 ~ 3h.In detection sensitivity, NASBA can detect the trace target lower than 10genecopies/ μ l in solution, and the detectability of PCR is at 100genecopies/ μ about l.Therefore, NASBA technology is that amplification efficiency or detection sensitivity are all higher than RT-PCR technology.And only needing the constant temperature of 41 DEG C due to the reaction of NASBA, water-bath can meet reaction requirement, and do not need the temperature control device that the Standard PCR such as complicated heating and cooling rely on, therefore the instrument cost of NASBA technology is very cheap.Be combined with corresponding detection technique, NASBA also has the plurality of advantages such as easy and simple to handle, high specificity.
Summary of the invention
First object of the present invention is to provide a kind of primer pair group for detecting parainfluenza virus.
Primer pair group for detecting parainfluenza virus provided by the present invention, is made up of following 3 primer pairs:
The primer pair 1 be made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2; The primer pair 2 be made up of two single strand dnas shown in sequence in sequence table 3 and sequence 4; The primer pair 3 be made up of two single strand dnas shown in sequence in sequence table 5 and sequence 6.
Wherein, described primer pair 1 (PIV1) is for the parainfluenza virus I type that increases; Described primer pair 2 (PIV2) is for the parainfluenza virus II type that increases; Described primer pair 3 (PIV3) is for the parainfluenza virus type III that increases.
Second object of the present invention is to provide a kind of complete single stranded DNA for detecting parainfluenza virus.
Complete single stranded DNA for detecting parainfluenza virus provided by the present invention, is made up of probe groups and described primer pair group; Described probe groups is made up of following 3 ssDNA probe: the ssDNA probe 1 in sequence table shown in sequence 7; SsDNA probe 2 in sequence table shown in sequence 8; SsDNA probe 3 in sequence table shown in sequence 9.
Wherein, described ssDNA probe 1 (PIV1_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 1; Described ssDNA probe 2 (PIV2_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 2; Described ssDNA probe 3 (PIV3_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 3.
5 ' end of the every bar ssDNA probe in described probe groups is all marked with fluorescent reporter group FAM, and 3 ' end is all marked with fluorescent quenching group TAMRA.
In described complete single stranded DNA provided by the present invention, the ssDNA probe of each primer pair and correspondence thereof can be packaged in separately in a packaging.As described in primer pair 1 and as described in ssDNA probe 1 be packaged in first packaging in; Described primer pair 2 and described ssDNA probe 2 are packaged in second packaging; Described primer pair 3 and described ssDNA probe 3 are packaged in the 3rd packaging.
3rd object of the present invention is to provide a kind of test kit for detecting parainfluenza virus.
Test kit for detecting parainfluenza virus provided by the present invention, containing described primer pair group or described complete single stranded DNA.
Described test kit also can contain internal reference primer pair and internal reference probe.Described internal reference primer pair is the primer pair GAPD_IC for house-keeping gene GAPDHmRNA in the human genome that increases, and is made up of two single strand dnas shown in sequence in sequence table 10 and sequence 11.Described internal reference probe (GAPD_IC_MB) is the ssDNA probe shown in sequence in sequence table 12, for the amplification of internal reference primer pair (GAPD_IC) described in Real-Time Monitoring.
5 ' end of described internal reference probe is marked with fluorescent reporter group FAM, and 3 ' end is marked with fluorescent quenching group TAMRA.
Also constant-temperature amplification damping fluid and constant-temperature amplification enzyme solution can be contained in described test kit.
The solvent of described constant-temperature amplification damping fluid is water, solute and concentration as follows: the Tris-HCL of 200mMpH8.0,50mMDTT, 10mMdNTP, 10mMrNTP, 80mMMgCl
2, 450mMKCl, 15% volumn concentration DMSO, 1M sorbyl alcohol, 20mM tetramethyl ammonium chloride.
The solvent of described constant-temperature amplification enzyme solution is water, solute and concentration as follows: AMV reversed transcriptive enzyme 1U/ μ l, t7 rna polymerase 5U/ μ l, ribonuclease H 0.5U/ μ l, Pyrophosphate phosphohydrolase 0.5U/ μ l, RNA enzyme inhibitors 5U/ μ l, BSA0.5 μ g/ μ l.
Also dish-style chip can be contained, as 24 reaction chamber dish-style chips in described test kit.
In the present invention, described 24 reaction chamber dish-style chips are the supporting dish-style chip of " the brilliant core RTisochipTM-A constant-temperature amplification micro-fluidic chip foranalysis of nucleic acids instrument " that Capitalbio Corporation Co., Ltd. produces, and its model is 1 × 24.
1# to the 5# reaction chamber of described dish-style chip deposits dry following (1)-(5) respectively:
(1) described primer pair 1 and described ssDNA probe 1; (2) described primer pair 2 and described ssDNA probe 2; (3) described primer pair 3 and described ssDNA probe 3; (4) described internal reference primer pair and described internal reference probe; (5) negative controls.
Described negative controls specifically can be the water (Rnase-free water) without RNA enzyme.
Wherein, method primer and probe being embedded into dish-style chip is: primer, the probe corresponding with described primer, agarose are mixed, be mixed with mixing solutions, make described primer, described probe and the final concentration of described agarose in described mixing solutions be respectively 0.2 μM, 50nM, 0.1% (mass percentage); Get mixing solutions described in 1 μ l and click and enter corresponding dish-style chip reaction chamber, after drying in clean super clean bench, compressing tablet encapsulates, and makes after punching press vacuumizes.
Described primer pair group or complete single stranded DNA or test kit are detecting or non-diagnostic object application in auxiliary detection parainfluenza virus also belongs to protection scope of the present invention.
In the application, by constant-temperature amplification enzyme solution described in constant-temperature amplification damping fluid described in 15 μ l and 10 μ l, be injected in described dish-style chip after mixing with 25 μ l sample to be tested solution, isothermal reaction 1h at 41 DEG C.
In the application, can Nasopharyngeal swabs as sample to be tested; Accordingly, described sample to be tested solution can be the RNA extracted from Nasopharyngeal swabs.
In the present invention, described parainfluenza virus specifically can be at least one as follows: parainfluenza virus I type, parainfluenza virus II type and parainfluenza virus type III.
Test kit provided by the invention supports high-throughput, parainfluenza virus infection can be detected quickly and accurately, for clinical, the detected result that can obtain 3 kinds of parainfluenza virus indexs in 1 hour not only faster than the real time fluorescence quantifying PCR method comparatively generally adopted at present, and for quick auxiliary direction treatment and medication also significant.Meanwhile, the detection of multi objective also may be used for regional epidemiological survey and epidemic situation monitoring, to study the popularity of parainfluenza virus infection in China.
Accompanying drawing explanation
Fig. 1 is dish-style chip and primer sample application cavity schematic diagram.
Fig. 2 is 3 kinds of parainfluenza virus reference material dish-style chip detection result figure.Wherein, A is the detected result of the recombinant plasmid pUC19-PIV1 containing parainfluenza virus I type target gene at 1# reaction chamber; B is the detected result of the recombinant plasmid pUC19-PIV2 containing parainfluenza virus II type target gene at 2# reaction chamber; C is the detected result of the recombinant plasmid pUC19-PIV3 containing parainfluenza virus type III target gene at 3# reaction chamber.In A-C, E1 represents that template is 10
1copy/μ l, by that analogy, E5 represents that template is 10
5copy/μ l; NC represents negative control.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1, for detecting preparation and the use thereof of the test kit of parainfluenza virus
One, for detecting the preparation of the test kit of parainfluenza virus
Test kit for detecting parainfluenza virus provided by the present invention is composed as follows:
1, constant-temperature amplification damping fluid
The solvent of constant-temperature amplification damping fluid is water, solute and concentration as follows: 200mMTris-HCL (pH8.0), 50mMDTT, 10mMdNTP, 10mMrNTP, 80mMMgCl
2, 450mMKCl, 15% volumn concentration DMSO, 1M sorbyl alcohol, 20mM tetramethyl ammonium chloride.
2, constant-temperature amplification enzyme solution
The solvent of constant-temperature amplification enzyme solution is water, solute and concentration as follows: AMV reversed transcriptive enzyme 1U/ μ l, t7 rna polymerase 5U/ μ l, ribonuclease H 0.5U/ μ l, Pyrophosphate phosphohydrolase 0.5U/ μ l, RNA enzyme inhibitors 5U/ μ l, BSA0.5 μ g/ μ l.
3,24 reaction chamber dish-style chips of primer pair and ssDNA probe are mounted with
Described 24 reaction chamber dish-style chips are the supporting dish-style chip of " the brilliant core RTisochipTM-A constant-temperature amplification micro-fluidic chip foranalysis of nucleic acids instrument " that Capitalbio Corporation Co., Ltd. produces, and its model is 1 × 24, and schematic diagram as shown in Figure 1.
1# to the 5# reaction chamber of described 24 reaction chamber dish-style chips deposits dry following (1)-(5) respectively:
(1) primer pair 1 and ssDNA probe 1; (2) primer pair 2 and ssDNA probe 2; (3) primer pair 3 and ssDNA probe 3; (4) internal reference primer pair and internal reference probe; (5) negative controls.Described negative controls is specially the water (Rnase-free water) without RNA enzyme.
Wherein, described primer pair 1 (PIV1), for the parainfluenza virus I type that increases, is made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2; Described primer pair 2 (PIV2), for the parainfluenza virus II type that increases, is made up of two single strand dnas shown in sequence in sequence table 3 and sequence 4; Described primer pair 3 (PIV3), for the parainfluenza virus type III that increases, is made up of two single strand dnas shown in sequence in sequence table 5 and sequence 6.Described internal reference primer pair (GAPD_IC), for house-keeping gene GAPDHmRNA in the human genome that increases, is made up of two single strand dnas shown in sequence in sequence table 10 and sequence 11.
Described ssDNA probe 1 (PIV1_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 1, and its nucleotides sequence is classified as sequence 7 in sequence table; Described ssDNA probe 2 (PIV2TYMB) is for the amplification of primer pair described in Real-Time Monitoring 2, and its nucleotides sequence is classified as sequence 8 in sequence table; Described ssDNA probe 3 (PIV3_TY_MB) is for the amplification of primer pair described in Real-Time Monitoring 3, and its nucleotides sequence is classified as sequence 9 in sequence table.Described internal reference probe (GAPD_IC_MB) is for the amplification of internal reference primer pair (GAPD_IC) described in Real-Time Monitoring, and its nucleotides sequence is classified as sequence 12 in sequence table.5 ' end of ssDNA probe described in every bar and described internal reference probe (GAPD_IC_MB) is all marked with fluorescent reporter group FAM, and 3 ' end is all marked with fluorescent quenching group TAMRA.
Wherein, method primer and probe being embedded into dish-style chip is: primer, the probe corresponding with described primer, agarose are mixed, be mixed with mixing solutions, make described primer, described probe and the final concentration of described agarose in described mixing solutions be respectively 0.2 μM, 50nM, 0.1% (mass percentage); Get mixing solutions described in 1 μ l and click and enter corresponding dish-style chip reaction chamber, after drying in clean super clean bench, compressing tablet encapsulates, and makes after punching press vacuumizes.
Two, for detecting the using method of the test kit of parainfluenza virus
1, reaction system preparation
Get 15 μ l constant-temperature amplification damping fluids (see step one 1), 10 μ l constant-temperature amplification enzyme solution (see step one 2), 25 μ l sample to be tested liquid are mixed into 50 μ l reaction solns, the 24 reaction chamber dish-style chips (see step one 3) being mounted with primer pair and ssDNA probe are injected, Quick spin 30s after sealing membrana oralis after vortex concussion evenly.
2, isothermal amplification reactions and detection
Dish-style chip is placed in rich Isochip-RT constant-temperature amplification instrument difficult to understand, 41 DEG C of reactions 1 hour is set, and complete real-time fluorescence scanning simultaneously.
3, result judges
After reaction terminates, according to the amplification curve of sample in each reaction chamber, determine the genome whether containing corresponding parainfluenza virus in described sample to be tested liquid as follows: if described testing sample produces S type amplification curve, then the genome containing corresponding parainfluenza virus in described sample to be tested liquid; Otherwise, then the genome not containing corresponding parainfluenza virus in described sample to be tested liquid.
Embodiment 2, for detecting sensitivity and the specificity analyses of the test kit of parainfluenza virus
One, the preparation of reference material RNA nucleic acid
1, the plasmid including parainfluenza virus target gene is built
(1) plasmid containing parainfluenza virus I type target gene
Between the multiple clone site EcoR V 464 ~ 1296 sections (Genbank SequenceID:gb|JF416791.1|, the UpdateDate:2014-12-31 of parainfluenza virus I type target gene sequence) of parainfluenza virus I type target gene sequence being inserted into pUC19 carrier (sky with biochemical corp's product).Obtain recombinant plasmid pUC19-PIV1.
(2) plasmid containing parainfluenza virus II type target gene
Between the multiple clone site EcoR V 2568 ~ 3629 sections (Genbank SequenceID:gb|DQ072586.1|, the UpdateDate:2008-9-4 of parainfluenza virus II type target gene sequence) of parainfluenza virus II type target gene sequence being inserted into pUC19 carrier (sky with biochemical corp's product).Obtain recombinant plasmid pUC19-PIV2.
(3) plasmid containing parainfluenza virus type III target gene
Between the multiple clone site EcoR V 211 ~ 1190 sections (Genbank SequenceID:dbj|AB623488.1|, the UpdateDate:2014-7-26 of parainfluenza virus type III target gene sequence) of parainfluenza virus type III target gene sequence being inserted into pUC19 carrier (sky with biochemical corp's product).Obtain recombinant plasmid pUC19-PIV3.
2, the preparation of reference material RNA nucleic acid
For examination recombinant plasmid: 3 kinds of step 1 structure contain the recombinant plasmid of corresponding parainfluenza virus target gene.
(1) enzyme is cut: first cut 2h by each for examination recombinant plasmid EcoRI restriction endonuclease 37 DEG C of enzymes.
(2) transcribe: by 5 μ l5 × TranscriptionOptimizedBuffer (Promega), 2UT7RNA polysaccharase, 10mMDTT (Promega), 10U recombinant RNA enzyme inhibitors (Promega), 2mMrNTP, it is the reaction system of 50 μ l that digestion products 5 μ l prepares cumulative volume, vibrations evenly latter 37 DEG C transcribe 4h.
(3) digest: the DNA digestive ferment (rDnaseI, the 5U/ μ l) system after having transcribed being added 1 μ l, shake centrifugal, hatch 20min for 37 DEG C.
(4) purifying: use RNA purification kit product (its catalog number is 740948) the purifying transcription product RNA:a as follows of Macherey-Nagel company) prepare RA1-C
2h
5oH mixed solution: with RA1:C
2h
5the proportions of OH=1:1 (volume ratio).In the transcription product of every 100 μ l, need to add 600 μ lRA1-C
2h
5oH mixed solution, i.e. 300 μ lRA1+300 μ lC
2h
5oH solution, here need according to the pipe number of transcription product calculate join the volume of mixed solution.If product is less than 100 μ l, then the amount of product is mended to 100 μ l with water and (in every pipe product, namely add the sky root Rnase-freeH of 50 μ l
2o).B) ready RA1-C is before incited somebody to action
2h
5oH mixed solution is assigned in 1.5ml centrifuge tube, two pipes, and 100 μ l products are transferred in corresponding centrifuge tube by often pipe 600 μ l, totally 700 μ l in pipe, and fully concussion is centrifugal.C) prepare two adsorption columns, and carry out corresponding mark, be transferred to by 700 μ l product mixture (adsorption column maximum capacity is 700 μ l) in adsorption column, the centrifugal 2min of 1.2 ten thousand rpm, outwells lower floor's solution.D) in adsorption column, add the RA3 of 700 μ l, place 1min, make it can be dispersed in the bottom of adsorption column fully, the centrifugal 2min of 1.2 ten thousand rpm, outwells lower floor's solution.E) in adsorption column, add the RA3 of 350 μ l, place the centrifugal 2min of 2min, 1.2 ten thousand rpm, outwell lower floor's solution.F) in adsorption column, add the RA3 of 300 μ l, place the centrifugal 2min of 2min, 1.2 ten thousand rpm, outwell lower floor's solution.G) after opening the lid 3min of adsorption column, 1.2 ten thousand rpm are empty from 2min, outwell lower floor's solution, repeat this step once.H) sky is after end, and adsorption column is transferred in corresponding centrifuge tube, opens adsorption column lid and places 15min, ethanol is volatilized completely, adds 60 μ lRnase-H in adsorption column
2o (carrying in test kit) wash-out, places the centrifugal 2min of 2min, 1.2 ten thousand rpm.I) centrifugal gained template is sucked back in adsorption column, place the centrifugal 2min of 2min, 1.2 ten thousand rpm, repeat this step twice.J) lose adsorption column, the template in centrifuge tube is taken out 2 μ l, measure its concentration with Nanodrop, and record its concentration and 260/280,260/230 ratio.
Two, for detecting sensitivity and the specificity analyses of the test kit of parainfluenza virus
1, the reference material Template preparation of different concns
By each RNA template (the 3 kinds of recombinant plasmids built in corresponding step one 1) all calculation in quantities to 10 after above-mentioned steps one purifying
10copy/μ l, and gradient dilution, obtain 10
5copy/μ l, 10
4copy/μ l, 10
3copy/μ l, 10
2the template dilution of the different concns such as copy/μ l, 10 copy/μ l.
2, reaction system preparation
Get 15 μ l constant-temperature amplification damping fluids (see embodiment 1 step 1), 10 μ l constant-temperature amplification enzyme solution (see embodiment 1 step 2), 25 μ l concentration template dilution be mixed into 50 μ l reaction solns, the 24 reaction chamber dish-style chips (see embodiment 1 step 3) being mounted with primer pair and ssDNA probe are injected, Quick spin 30s after sealing membrana oralis after vortex concussion evenly.
3, isothermal amplification reactions and detection
Dish-style chip is placed in rich Isochip-RT constant-temperature amplification instrument difficult to understand, 41 DEG C of reactions 1 hour is set, and complete real-time fluorescence scanning simultaneously.And according to the method for embodiment 1 step 23, result is judged.
Three, result
Result is as shown in Figure 2:
(1) for the RNA sample of recombinant plasmid pUC19-PIV1, the 1# reaction chamber of 24 reaction chamber dish-style chips is to 10
5copy/μ l, 10
4copy/μ l, 10
3copy/μ l, 10
2the detected result of copy/μ l, 10 copy/μ l templates all has obvious S type amplification curve, is shown as the positive; And 2# and 3# reaction chamber is all without amplification curve, be shown as feminine gender.
(2) for the RNA sample of recombinant plasmid pUC19-PIV2, the 2# reaction chamber of 24 reaction chamber dish-style chips is to 10
5copy/μ l, 10
4copy/μ l, 10
3copy/μ l, 10
2the detected result of copy/μ l, 10 copy/μ l templates all has obvious S type amplification curve, is shown as the positive; And 1# and 3# reaction chamber is all without amplification curve, be shown as feminine gender.
(3) for the RNA sample of recombinant plasmid pUC19-PIV3, the 3# reaction chamber of 24 reaction chamber dish-style chips is to 10
5copy/μ l, 10
4copy/μ l, 10
3copy/μ l, 10
2the detected result of copy/μ l, 10 copy/μ l templates all has obvious S type amplification curve, is shown as the positive; And 1# and 2# reaction chamber is all without amplification curve, be shown as feminine gender.
In addition, for the RNA sample of all 3 kinds of recombinant plasmids, the 4# reaction chamber (internal reference) of 24 reaction chamber dish-style chips and 5# reaction chamber (negative control) are all without amplification curve.Why 4# reaction chamber (internal reference) is because sample to be tested is the RNA nucleic acid of 3 kinds of recombinant plasmids without amplification curve, the RNA not containing house-keeping gene GAPDH in human genome.
These results suggest that this test kit has higher amplification sensitivity and specific amplification.
The detection of embodiment 3, actual clinical sample
One, clinical sample type
The present embodiment adopt clinical sample from Shenzhen San Yuan in the Nasopharyngeal swabs sample (in line with the principle that this picker is voluntary) gathered, swab of adopting is deposited in 3mL physiological saline, and totally 560 is routine.
Two, the extraction of viral nucleic acid in clinical sample
It is QIAampViralRNAMiniKit (Qiagen) that clinical sample viral nucleic acid extracts test kit used, extracts as follows:
(1) get in step one deposit clinical sample swab physiological saline 140 μ l in 1.5ml centrifuge tube;
(2) add 560 μ l to contain the BufferAVL of CarrierRNA mixed solution (namely 5.6 μ lCarrierRNA mixed solution+560 μ lBufferAVL are in centrifuge tube, slight whirlpool 15s;
(3) after brief centrifugation completes, room temperature (15-25 DEG C) places 10-20min, to ensure that BufferAVL has the sufficient time to carry out cracking in centrifuge tube;
(4) 560 μ l ethanol (96-100%, volumn concentration) are added in centrifuge tube, whirlpool mixing 15s, centrifugal;
(5) get 630 μ l sample solutions in adsorption column, place 2min, make it fully contact with adsorption column, 8000rpm, centrifugal 1min, change clean collection tube;
(6) if sample solution is more than 630 μ l, then previous step is repeated;
(7) in adsorption column, add the AW1 washing lotion of 500 μ l, place 2min, make it fully contact with adsorption column, the centrifugal 1min of 8000rpm, changes clean collection tube;
(8) in adsorption column, add the AW2 washing lotion of 500 μ l, place 2min, make it fully contact with adsorption column, the centrifugal 3min of 12000rpm, changes clean collection tube;
(9) in adsorption column, add the AW2 washing lotion of 300 μ l, place 2min, make it fully contact with adsorption column, the centrifugal 3min of 12000rpm, changes clean collection tube;
(10) adsorption column lid is opened placement 2min, make pressure inside and outside it consistent, 8000rpm is empty from 1min.Empty after end, repeat this step once.
(11) sky is after end, is transferred to by adsorption column in 1.5ml centrifuge tube, opens adsorption column lid, places 20-30min, makes ethanol volatilize completely;
(12) add 50 μ lBufferAVE wash-out nucleic acid, the centrifugal 1min of 8000rpm, centrifugal complete after, the nucleic acid under wash-out is sucked back adsorption column, after centrifugal 1min, loses adsorption column.
Three, the detection of actual clinical sample
1, reaction system preparation
Get 15 μ l constant-temperature amplification damping fluids (see embodiment 1 step one 1), 10 μ l constant-temperature amplification enzyme solution (see embodiment 1 step one 2), 25 μ l step 2 extract the clinical sample solution obtained and be mixed into 50 μ l reaction solns, the 24 reaction chamber dish-style chips (see embodiment 1 step one 3) being mounted with primer pair and ssDNA probe are injected, Quick spin 30s after sealing membrana oralis after vortex concussion evenly.
2, isothermal amplification reactions and detection
Dish-style chip is placed in rich Isochip-RT constant-temperature amplification instrument difficult to understand, 41 DEG C of reactions 1 hour is set, and complete real-time fluorescence scanning simultaneously.And according to the method for embodiment 1 step 23, result is judged.
Experiment arranges the reliability demonstration of conventional RT-PCR method for test kit detected result of the present invention simultaneously.Specificity RT-PCR primer sequence for detecting each virus is as shown in table 1.
Table 1 is for detecting the specificity RT-PCR primer sequence of 3 kinds of parainfluenza viruses
| Index name | RT-PCR upstream primer (5 '-3 ') | RT-PCR downstream primer (5 '-3 ') |
| Parainfluenza virus I type | GTAGTCTCATTCACAGTGGGYAAGGA | ATCTCATTATTACCYGGACCAAGTCT |
| Parainfluenza virus II type | TACAAGACACAACCTCCTGGTATAGCA | GGACGCCTAAATATGGACCTCTCCT |
| Parainfluenza virus type III | TCTCTCTGTGTTTTCCCVGGACACCC | GACTTAAATCCYAGGATCTCTCATAC |
Four, result
The detected result display of test kit of the present invention, for each parainfluenza virus, adopts the sample example of the RT-PCR method detection positive to adopt test kit of the present invention to detect and all shows positive findings.In addition, for a few sample example, adopt RT-PCR method to detect negative, but adopt test kit detected result of the present invention to be positive.Detecting by carrying out later stage resampling RT-PCR to these sample examples, finding that these sample examples are detected as corresponding parainfluenza virus really positive.More than show, test kit of the present invention is to the recall rate of corresponding parainfluenza virus higher than RT-PCR method, and its detected result accurately and reliably.Test kit of the present invention and RT-PCR method detect the statistics of parainfluenza virus clinical sample see table 2.
Table 2 test kit of the present invention and RT-PCR method detect the statistics of parainfluenza virus clinical sample
Claims (10)
1., for detecting the primer pair group of parainfluenza virus, be made up of following 3 primer pairs:
The primer pair 1 be made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2;
The primer pair 2 be made up of two single strand dnas shown in sequence in sequence table 3 and sequence 4;
The primer pair 3 be made up of two single strand dnas shown in sequence in sequence table 5 and sequence 6.
2., for detecting the complete single stranded DNA of parainfluenza virus, be made up of probe groups and primer pair group according to claim 1;
Described probe groups is made up of following 3 ssDNA probe: the ssDNA probe 1 in sequence table shown in sequence 7; SsDNA probe 2 in sequence table shown in sequence 8; SsDNA probe 3 in sequence table shown in sequence 9.
3. complete single stranded DNA according to claim 2, is characterized in that: 5 ' end of the every bar ssDNA probe in described probe groups is all marked with fluorescent reporter group FAM, and 3 ' end is all marked with fluorescent quenching group TAMRA.
4. for detecting the test kit of parainfluenza virus, containing the complete single stranded DNA described in primer pair group according to claim 1 or Claims 2 or 3.
5. test kit according to claim 4, is characterized in that: described test kit is also containing internal reference primer pair and internal reference probe; Described internal reference primer pair is made up of two single strand dnas shown in sequence in sequence table 10 and sequence 11; Described internal reference probe is the ssDNA probe shown in sequence in sequence table 12;
Concrete, 5 ' end of described internal reference probe is marked with fluorescent reporter group FAM, and 3 ' end is marked with fluorescent quenching group TAMRA.
6. the test kit according to claim 4 or 5, is characterized in that: also containing constant-temperature amplification damping fluid and constant-temperature amplification enzyme solution in described test kit;
The solvent of described constant-temperature amplification damping fluid is water, solute and concentration as follows: the Tris-HCL of 200mMpH8.0,50mMDTT, 10mMdNTP, 10mMrNTP, 80mMMgCl
2, 450mMKCl, 15% volumn concentration DMSO, 1M sorbyl alcohol, 20mM tetramethyl ammonium chloride;
The solvent of described constant-temperature amplification enzyme solution is water, solute and concentration as follows: AMV reversed transcriptive enzyme 1U/ μ l, t7 rna polymerase 5U/ μ l, ribonuclease H 0.5U/ μ l, Pyrophosphate phosphohydrolase 0.5U/ μ l, RNA enzyme inhibitors 5U/ μ l, BSA0.5 μ g/ μ l.
7. according to described test kit arbitrary in claim 4-6, it is characterized in that: also containing dish-style chip in described test kit;
1# to the 5# reaction chamber of described dish-style chip deposits dry following (1)-(5) respectively:
(1) primer pair 1 described in claim 1 and the ssDNA probe described in claim 21;
(2) primer pair 2 described in claim 1 and the ssDNA probe described in claim 22;
(3) primer pair 3 described in claim 1 and the ssDNA probe described in claim 23;
(4) the internal reference primer pair described in claim 5 and described internal reference probe;
(5) negative controls.
8. in claim 1-7 arbitrary described primer pair group or complete single stranded DNA or test kit in the non-diagnostic object application detected or in auxiliary detection parainfluenza virus.
9. application according to claim 8, it is characterized in that: in described application, by constant-temperature amplification enzyme solution described in constant-temperature amplification damping fluid described in 15 μ l and 10 μ l, be injected in described dish-style chip after mixing with 25 μ l sample to be tested solution, isothermal reaction 1h at 41 DEG C.
10., according to arbitrary described primer pair group in 1-9 or complete single stranded DNA or test kit or application, it is characterized in that: described parainfluenza virus be following at least one: parainfluenza virus I type, parainfluenza virus II type and parainfluenza virus type III.
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| CN109207640A (en) * | 2018-10-23 | 2019-01-15 | 深圳市亿立方生物技术有限公司 | It is a kind of detect various respiratory road virus primer sets, probe groups and kit and its application |
| CN111621585A (en) * | 2020-04-16 | 2020-09-04 | 大连民族大学 | Rapid detection kit for simultaneously detecting multiple transgenic rape lines and application thereof |
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