CN105018648B - A kind of kit and its application for being used to detect Respirovirus - Google Patents
A kind of kit and its application for being used to detect Respirovirus Download PDFInfo
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Abstract
The invention discloses a kind of kit and its application for being used to detect Respirovirus.Contain the primer pair group for being used for detecting Respirovirus in kit provided by the present invention, be made up of 16 primer pairs, its sequence is sequence 1 32 in sequence table.Kit provided by the invention supports high flux, common respiratory virus infection can quickly and accurately be detected, for clinic, the testing result that 16 kinds of viral indexs can be obtained in 1 hour is not only faster than the real time fluorescence quantifying PCR method more generally used at present, and also significant for the treatment of quick auxiliary direction and medication.Meanwhile the detection of multi objective can be used for regional epidemiological survey and epidemic situation monitoring, to study popularity of the respiratory virus infection in China.
Description
Technical field
The invention belongs to nucleic acid amplification technologies field, is related to a kind of kit for being used to detect Respirovirus and its answers
With.
Background technology
Respiratory tract infection is divided into the infection of the upper respiratory tract and ALRI, and main pathogens include bacterium, virus, Zhi Yuan
Body, Chlamydia, fungi etc., wherein mainly based on viral infection, have more than 80% in particularly upper breathing respiratory tract infection
It is viral.The sick four seasons, any age can fall ill, and be entered by the apparatus containing the virulent spittle, droplet, or through pollution
Row is propagated.Often when Abwehrkraft des Koepers reduces, situations such as such as catching cold, be tired, drenching with rain, virus former existing or by external world's intrusion
Or bacterium, breeding is mushroomed out, causes to infect, is grown up general 5-7 days and fully recovers.
Childhood infection respiratory pathogen generally induces ARI (ARI), and ARI is that infant, children are common
Disease, Chang Jifa bronchitis, pneumonia, paranasal sinusitis, minority can complicated with acute meningitis, myocarditis, ephritis, rheumatic fever etc.,
Complication seriously endangers the health of children.95% ARI is had proven to caused by the pathogen beyond bacterium, virus is ARI
Main pathogens.Children in different ages is otherwise varied to virus susceptibility, is shown according to WHO data, and respiratory tract infection is world's model
Enclose interior underage child (<5 years old) dead the first reason, about 5,000,000 death of child can be caused every year.Children with acute respiratory tract infection
Ill 3-8 times/year of number average out to, in the ratio that 1 years old infects in children about 1~3%, school-ager to 5%~10%.By
Be not quite similar in popular viral species of each period and type, sometimes a few different virus again can mixed infection, it is therefore right
The monitoring of childrens respiratory tract virus causing disease has very important meaning.Rapid＆Early diagnosis respiratory virus infection can be timely
Guiding clinical treatment provides foundation for reasonable selection antiviral antibacterial medicine and had in terms of abuse of antibiotics is prevented important
Meaning.
Respirovirus refers to the virus for infecting respiratory tract or being propagated by respiratory tract, refers at least to 8 sections,
The virus of more than 200 kinds of type.Clinical common Flu-A of the virus including orthomyxoviridae family for causing respiratory tract infection is sick
Malicious (InfA) and influenza B virus (InfB), parainfluenza virus 1-3 types (PIV1-PIV3), the respiratory tract of Paramyxoviridae close
Cellular virus (RSV), human metapneumovirus (hMPV), the adenovirus (AdV) of Adenoviridae, the rhinovirus of Pironavirus section
(HRV), coronavirus (Corn) and enterovirus (Entv) etc..The method for detecting common Respirovirus at present is more, but
All Shortcomings, such as Electronic Speculum detection method complex and expensive, virus purification culture is time-consuming extremely long and false negative is more, is taking culture
As a result clinical meaning has generally been lost after, ELISA detection is special viral antibody but is once only capable of detecting a kind of disease
Poison.And the detection of nucleic acids based on nucleic acid amplification, due to speed fast (can complete to detect in usual 3 hours), high sensitivity, specificity
It is good, it is increasingly becoming goldstandard in field of virus detection.
The amplification technique (Nucleic Acid Sequence Based Amplification) of nucleotide sequence is relied on, i.e.,
NASBA amplification techniques, it is to be mediated by pair of primers, be specific in vitro and continuous homogeneous to single stranded RNA progress constant temperature expansion
The process of increasing.Under 41 DEG C of constant temperatures, in terms of reaction speed, template ribonucleic acid can be expanded about 10 by NASBA amplifications 1h9~1012
Times, and common PCR reactions need 2~3h.In terms of detection sensitivity, NASBA, which can be detected, is less than 10gene in solution
Copies/ μ l trace target, and PCR test limit is in 100gene copies/ μ l or so.Therefore, NASBA technologies are either
Amplification efficiency or detection sensitivity are all higher than RT-PCR technology.And because NASBA reaction only needs one 41 DEG C of perseverance
Warm condition, water-bath can meet that reaction requires, it is not necessary to the temperature control device that the Standard PCR such as complicated heating and cooling is relied on, because
The instrument cost of this NASBA technology is very cheap.Combined with corresponding detection technique, NASBA also has easy to operate, specific
Many advantages, such as strong.
The content of the invention
First purpose of the present invention is to provide a kind of primer pair group for being used to detect Respirovirus.
The primer pair group provided by the present invention for being used to detect Respirovirus, is made up of following 16 primer pairs：
The primer pair 1 being made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2；By sequence in sequence table
The primer pair 2 of two single strand dnas composition shown in row 3 and sequence 4；As two shown in sequence in sequence table 5 and sequence 6
The primer pair 3 of single strand dna composition；Drawn by what two single strand dnas shown in sequence in sequence table 7 and sequence 8 formed
Thing is to 4；The primer pair 5 being made up of two single strand dnas shown in sequence in sequence table 9 and sequence 10；By sequence in sequence table
The primer pair 6 of two single strand dnas composition shown in row 11 and sequence 12；As shown in sequence in sequence table 13 and sequence 14
The primer pair 7 of two single strand dna compositions；As two single strand dna groups shown in sequence in sequence table 15 and sequence 16
Into primer pair 8；The primer pair 9 being made up of two single strand dnas shown in sequence in sequence table 17 and sequence 18；By sequence
Sequence 19 and the primer pair 10 of two single strand dnas composition shown in sequence 20 in table；By sequence in sequence table 21 and sequence
The primer pair 11 of two single strand dnas composition shown in 22；It is single-stranded as two shown in sequence in sequence table 23 and sequence 24
The primer pair 12 of DNA molecular composition；The primer being made up of two single strand dnas shown in sequence in sequence table 25 and sequence 26
To 13；The primer pair 14 being made up of two single strand dnas shown in sequence in sequence table 27 and sequence 28；By sequence in sequence table
The primer pair 15 of two single strand dnas composition shown in row 29 and sequence 30；As shown in sequence in sequence table 31 and sequence 32
Two single strand dnas composition primer pair 16.
Wherein, the primer pair 1 (InfA_TY) is used to expand influenza A virus；The primer pair 2 (InfA_H1) is used
In amplification influenza A virus H1 hypotypes；The primer pair 3 (InfA_H3) is used to expand influenza A virus H3 hypotypes；It is described
Primer pair 4 (InfB_TY) is used to expand influenza B virus；The primer pair 5 (RSV_TY) is used to expand respiratory syncystial disease
Poison；The primer pair 6 (HRV_TY) is used to expand rhinovirus；The primer pair 7 (ADV_TY) is used to expand adenovirus；It is described to draw
Thing is used to expand parainfluenza virus I types to 8 (PIV1)；The primer pair 9 (PIV2) is used to expand parainfluenza virus II types；It is described
Primer pair 10 (PIV3) is used to expand parainfluenza virus type III；The primer pair 11 (Corn_OC) is used to expand coronavirus
HKU1/OC43；The primer pair 12 (Corn_NL) is used to expand HCoV-229E/NL63；(the hMPV_ of primer pair 13
TY) it is used to expand human metapneumovirus；The primer pair 14 (Entv_TY) is used to expand enterovirus；The primer pair 15
(EV71_VP1) it is used to expand enterovirus EV 71 type；The primer pair 16 (CA16_VP1) is used to expand enterovirus CA16
Type.
Second object of the present invention is to provide a kind of complete single stranded DNA for being used to detect Respirovirus.
The complete single stranded DNA provided by the present invention for being used to detect Respirovirus, by probe groups and the primer pair group
Composition；The probe groups are made up of following 16 ssDNA probes：SsDNA probe 1 in sequence table shown in sequence 33；Sequence
SsDNA probe 2 in list shown in sequence 34；SsDNA probe 3 in sequence table shown in sequence 35；Sequence in sequence table
SsDNA probe 4 shown in 36；SsDNA probe 5 in sequence table shown in sequence 37；List in sequence table shown in sequence 38
Ssdna probe 6；SsDNA probe 7 in sequence table shown in sequence 39；SsDNA probe 8 in sequence table shown in sequence 40；
SsDNA probe 9 in sequence table shown in sequence 41；SsDNA probe 10 in sequence table shown in sequence 42；Sequence in sequence table
SsDNA probe 11 shown in row 43；SsDNA probe 12 in sequence table shown in sequence 44；In sequence table shown in sequence 45
SsDNA probe 13；SsDNA probe 14 in sequence table shown in sequence 46；Single stranded DNA in sequence table shown in sequence 47
Probe 15；SsDNA probe 16 in sequence table shown in sequence 48.
Wherein, the ssDNA probe 1 (InfA_TY_MB) is used for the amplification for monitoring the primer pair 1 in real time；Institute
State the amplification that ssDNA probe 2 (InfA_H1_MB) is used to monitor the primer pair 2 in real time；The ssDNA probe 3
(InfA_H3_MB) it is used for the amplification for monitoring the primer pair 3 in real time；The ssDNA probe 4 (InfB_TY_MB) is used
In the amplification for monitoring the primer pair 4 in real time；The ssDNA probe 5 (RSV_TY_MB) is used to draw described in monitoring in real time
Thing to 5 amplification；The ssDNA probe 6 (HRV_TY_MB) is used for the amplification for monitoring the primer pair 6 in real time；
The ssDNA probe 7 (ADV_TY_MB) is used for the amplification for monitoring the primer pair 7 in real time；The ssDNA probe 8
(PIV1_TY_MB) it is used for the amplification for monitoring the primer pair 8 in real time；The ssDNA probe 9 (PIV2_TY_MB) is used
In the amplification for monitoring the primer pair 9 in real time；The ssDNA probe 10 (PIV3_TY_MB) is used for described in monitoring in real time
The amplification of primer pair 10；The ssDNA probe 11 (Corn_OC_MB) is used for the expansion for monitoring the primer pair 11 in real time
Increase result；The ssDNA probe 12 (Corn_NL_MB) is used for the amplification for monitoring the primer pair 12 in real time；The list
Ssdna probe 13 (hMPV_TY_MB) is used for the amplification for monitoring the primer pair 13 in real time；The ssDNA probe 14
(Entv_TY_MB) it is used for the amplification for monitoring the primer pair 14 in real time；The ssDNA probe 15 (EV71_VP1_MB)
For monitoring the amplification of the primer pair 15 in real time；The ssDNA probe 16 (CA16_VP1_MB) is used to monitor in real time
The amplification of the primer pair 16.
5 ' the ends mark of every ssDNA probe in the probe groups has FAM, and 3 ' ends mark
There is fluorescent quenching group TAMRA.
In the complete single stranded DNA provided by the present invention, each primer pair and its corresponding ssDNA probe can be single
Solely it is packaged in a packaging.As the primer pair 1 and the ssDNA probe 1 are packaged in first packaging；The primer
2 and the ssDNA probe 2 are packaged in second packaging；The primer pair 3 and the ssDNA probe 3 are packaged in
In three packagings；The primer pair 4 and the ssDNA probe 4 are packaged in the 4th packaging；The primer pair 5 and described
SsDNA probe 5 is packaged in the 5th packaging；The primer pair 6 and the ssDNA probe 6 are packaged in the 6th packaging
It is interior；The primer pair 7 and the ssDNA probe 7 are packaged in the 7th packaging；The primer pair 8 and the single stranded DNA are visited
Pin 8 is packaged in the 8th packaging；The primer pair 9 and the ssDNA probe 9 are packaged in the 9th packaging；It is described to draw
Thing is packaged in 10 and the ssDNA probe 10 in the tenth packaging；The primer pair 11 and the ssDNA probe 1 are wrapped
In the 11st packaging；The primer pair 12 and the ssDNA probe 12 are packaged in the 12nd packaging；It is described to draw
Thing is packaged in 13 and the ssDNA probe 13 in the 13rd packaging；The primer pair 14 and the ssDNA probe 14
It is packaged in the 14th packaging；The primer pair 15 and the ssDNA probe 15 are packaged in the 15th packaging；It is described
Primer pair 16 and the ssDNA probe 16 are packaged in the 16th packaging.
Third object of the present invention is to provide a kind of kit for being used to detect Respirovirus.
The kit provided by the present invention for being used to detect Respirovirus, contains the primer pair group or the complete list
Chain DNA.
The kit can also contain internal reference primer pair and internal reference probe.The internal reference primer pair is for expanding
House-keeping gene GAPDH mRNA primer pair GAPD_IC in human genome, as two shown in sequence in sequence table 49 and sequence 50
Bar single strand dna forms.The internal reference probe (GAPD_IC_MB) is that the single stranded DNA in sequence table shown in sequence 51 is visited
Pin, for monitoring the amplification of the internal reference primer pair (GAPD_IC) in real time.
5 ' ends of the internal reference probe are marked with fluorescent reporter group FAM, and 3 ' ends are marked with fluorescent quenching group
TAMRA。
Constant-temperature amplification buffer solution and constant-temperature amplification enzyme solutions can also be contained in the kit.
The solvent of the constant-temperature amplification buffer solution is water, and solute and concentration are as follows：200mM pH 8.0 Tris-HCL,
50mM DTT, 10mM dNTP, 10mM rNTP, 80mM MgCl2, 450mM KCl, 15% volumn concentration DMSO, 1M sorb
Alcohol, 20mM tetramethyl ammonium chlorides.
The solvent of the constant-temperature amplification enzyme solutions is water, and solute and concentration are as follows：AMV reverse transcriptase 1U/ μ l, T7RNA gather
Synthase 5U/ μ l, ribonuclease H 0.5U/ μ l, the μ g/ μ l of pyrophosphatase 0.5U/ μ l, RNase inhibitor 5U/ μ l, BSA 0.5.
Dish-style chip can also be contained in the kit, such as 24 reaction chamber dish-style chips.
In the present invention, the 24 reaction chamber dish-style chip is " the brilliant core of Capitalbio Corporation Co., Ltd.'s production
The supporting dish-style chip of RTisochipTM-A constant-temperature amplification micro-fluidic chips nucleic acids instrument ", its model 1 × 24.
The 1# of the dish-style chip deposits dry following (1)-(18) to 18# reaction chambers respectively：
(1) primer pair 1 and the ssDNA probe 1；(2) primer pair 2 and the ssDNA probe 2；(3)
The primer pair 3 and the ssDNA probe 3；(4) primer pair 4 and the ssDNA probe 4；(5) primer pair 5
With the ssDNA probe 5；(6) primer pair 6 and the ssDNA probe 6；(7) primer pair 7 and described single-stranded
DNA probe 7；(8) primer pair 8 and the ssDNA probe 8；(9) primer pair 9 and the ssDNA probe 9；
(10) primer pair 10 and the ssDNA probe 10；(11) primer pair 11 and the ssDNA probe 11；(12)
The primer pair 12 and the ssDNA probe 12；(13) primer pair 13 and the ssDNA probe 13；(14) it is described
Primer pair 14 and the ssDNA probe 14；(15) primer pair 15 and the ssDNA probe 15；(16) primer
To 16 and the ssDNA probe 16；(17) the internal reference primer pair and the internal reference probe；(18) negative controls.
The negative controls concretely water without RNase (Rnase-free water).
Wherein, the method for primer and probe being embedded into dish-style chip is：By primer, the spy corresponding with the primer
Pin, agarose mixing, are configured to mixed solution, make the primer, the probe and the agarose in the mixed solution
Final concentration be respectively 0.2 μM, 50nM, 0.1% (weight/mass percentage composition)；Mixed solution described in 1 μ l is taken to click and enter corresponding dish-style
Chip reaction chamber, after being dried in clean super-clean bench, tabletting encapsulation, punching press is made after vacuumizing.
The primer pair group or complete single stranded DNA or kit are non-diagnostic in detecting or aiding in detection Respirovirus
Purpose application falls within protection scope of the present invention.
In the application, by constant-temperature amplification enzyme solutions described in constant-temperature amplification buffer solution described in 15 μ l and 10 μ l, with 25 μ l
It is injected into after the mixing of sample to be tested solution in the dish-style chip, isothermal reaction 1h at 41 DEG C.
In the application, sample to be tested can be used as using Nasopharyngeal swabs；Accordingly, the sample to be tested solution can be from nose
The RNA extracted in throat swab.
In the present invention, the Respirovirus is concretely at least one of following：Influenza A virus, A type stream
Influenza Virus H1 hypotypes, influenza A virus H3 hypotypes, influenza B virus, Respiratory Syncytial Virus(RSV), rhinovirus, adenovirus, pair
Influenza virus I types, parainfluenza virus II types, parainfluenza virus type III, HCoV-HKU1/OC43, HCoV-229E/
NL63, human metapneumovirus, enterovirus, enterovirus EV 71 type and enterovirus CA16 types.
Kit provided by the invention supports high flux, can quickly and accurately detect common respiratory virus infection, for
For clinic, the testing result that 16 kinds of viral indexs can be obtained in 1 hour is not only faster than the reality more generally used at present
When fluorescence quantifying PCR method, and for quick auxiliary direction treatment and medication it is also significant.Meanwhile multi objective
Detection can be used for regional epidemiological survey and epidemic situation monitoring, to study prevalence of the respiratory virus infection in China
Situation.
Brief description of the drawings
Fig. 1 is dish-style chip and primer sample application cavity schematic diagram.
Fig. 2 is 16 kinds of Respirovirus reference material dish-style chip testing result figures.Wherein, A is to contain influenza A virus
Testing results of the recombinant plasmid pUC19-InfA of MP target genes in 1# reaction chambers；B is to contain influenza A virus H1 hypotypes
Testing results of the recombinant plasmid pUC19-InfA_H1 of target gene in 2# reaction chambers；C is to contain influenza A virus H3 hypotypes
Testing results of the recombinant plasmid pUC19-InfA_H3 of target gene in 3# reaction chambers；D is to contain influenza B virus MP targets
Testing results of the recombinant plasmid pUC19-InfB of gene in 4# reaction chambers；E is to contain Respiratory Syncytial Virus(RSV) target gene
Testing results of the recombinant plasmid pUC19-RSV in 5# reaction chambers；F is the recombinant plasmid pUC19- containing rhinovirus target gene
Testing results of the HRV in 6# reaction chambers；G is the recombinant plasmid pUC19-ADV containing adenovirus target gene in 7# reaction chambers
Testing result；H is detection knots of the recombinant plasmid pUC19-PIV1 containing parainfluenza virus I type target genes in 8# reaction chambers
Fruit；I is testing results of the recombinant plasmid pUC19-PIV2 containing parainfluenza virus II type target genes in 9# reaction chambers；J is
Testing results of the recombinant plasmid pUC19-PIV3 containing parainfluenza virus type III target gene in 10# reaction chambers；K be containing
Testing results of the recombinant plasmid pUC19-Corn_OC of HCoV-HKU1/OC43 target genes in 11# reaction chambers；L be containing
Testing results of the recombinant plasmid pUC19-Corn_NL of HCoV-229E/NL63 target genes in 12# reaction chambers；M be containing
Testing results of the recombinant plasmid pUC19-hMPV of human metapneumovirus target gene in 13# reaction chambers；N is to contain enterovirus target
Mark testing results of the recombinant plasmid pUC19-Entv in 14# reaction chambers of gene；O is to contain enterovirus EV 71 target gene
Testing results of the recombinant plasmid pUC19-EV71 in 15# reaction chambers；P is the recombinant plasmid containing enterovirus CA16 target genes
Testing results of the pUC19-CA16 in 16# reaction chambers.In A-P, E1 represents that template is 101Copy/μ l, by that analogy, E5 represent
Template is 105Copy/μ l；NC represents negative control.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
The preparation and its use of embodiment 1, kit for detecting Respirovirus
First, for the preparation for the kit for detecting Respirovirus
Kit forms provided by the present invention for detecting Respirovirus are as follows：
1st, constant-temperature amplification buffer solution
The solvent of constant-temperature amplification buffer solution is water, and solute and concentration are as follows：200mM Tris-HCL (pH 8.0), 50mM
DTT, 10mM dNTP, 10mM rNTP, 80mM MgCl2, 450mM KCl, 15% volumn concentration DMSO, 1M sorbierite,
20mM tetramethyl ammonium chlorides.
2nd, constant-temperature amplification enzyme solutions
The solvent of constant-temperature amplification enzyme solutions is water, and solute and concentration are as follows：AMV reverse transcriptase 1U/ μ l, t7 rna polymerase
5U/ μ l, ribonuclease H 0.5U/ μ l, the μ g/ μ l of pyrophosphatase 0.5U/ μ l, RNase inhibitor 5U/ μ l, BSA 0.5.
3rd, 24 reaction chamber dish-style chips of primer pair and ssDNA probe are mounted with
The 24 reaction chamber dish-style chip is " the brilliant core RTisochipTM-A perseverances of Capitalbio Corporation Co., Ltd.'s production
The supporting dish-style chip of temperature amplification micro-fluidic chip nucleic acids instrument ", its model 1 × 24, schematic diagram is as shown in Figure 1.
The 1# of the 24 reaction chamber dish-style chip deposits dry following (1)-(18) to 18# reaction chambers respectively：
(1) primer pair 1 and ssDNA probe 1；(2) primer pair 2 and ssDNA probe 2；(3) primer pair 3 and single stranded DNA
Probe 3；(4) primer pair 4 and the DNA probe 4；(5) primer pair 5 and ssDNA probe 5；(6) primer pair 6 and single stranded DNA are visited
Pin 6；(7) primer pair 7 and ssDNA probe 7；(8) primer pair 8 and ssDNA probe 8；(9) primer pair 9 and ssDNA probe
9；(10) primer pair 10 and ssDNA probe 10；(11) primer pair 11 and ssDNA probe 11；(12) primer pair 12 and single-stranded
DNA probe 12；(13) primer pair 13 and ssDNA probe 13；(14) primer pair 14 and ssDNA probe 14；(15) primer pair
15 and ssDNA probe 15；(16) primer pair 16 and ssDNA probe 16；(17) internal reference primer pair and internal reference probe；
(18) negative controls.The negative controls are specially the water (Rnase-free water) without RNase.
Wherein, the primer pair 1 (InfA_TY) is used to expand influenza A virus, by sequence in sequence table 1 and sequence 2
Shown two single strand dnas composition；The primer pair 2 (InfA_H1) is used to expand influenza A virus H1 hypotypes, by sequence
Sequence 3 and two single strand dnas composition shown in sequence 4 in list；The primer pair 3 (InfA_H3) is used to expand A type
Influenza virus H3 hypotypes, it is made up of two single strand dnas shown in sequence in sequence table 5 and sequence 6；The primer pair 4
(InfB_TY) it is used to expand influenza B virus, as two single strand dna groups shown in sequence in sequence table 7 and sequence 8
Into；The primer pair 5 (RSV_TY) is used to expand Respiratory Syncytial Virus(RSV), as two shown in sequence in sequence table 9 and sequence 10
Bar single strand dna forms；The primer pair 6 (HRV_TY) is used to expand rhinovirus, by sequence in sequence table 11 and sequence 12
Shown two single strand dnas composition；The primer pair 7 (ADV_TY) is used to expand adenovirus, by sequence in sequence table 13
With two single strand dnas composition shown in sequence 14；The primer pair 8 (PIV1) is used to expand parainfluenza virus I types, by sequence
Sequence 15 and two single strand dnas composition shown in sequence 16 in list；The primer pair 9 (PIV2) is used to expand parainfluenza
Virus Type II, it is made up of two single strand dnas shown in sequence in sequence table 17 and sequence 18；The primer pair 10 (PIV3)
For expanding parainfluenza virus type III, it is made up of two single strand dnas shown in sequence in sequence table 19 and sequence 20；Institute
State primer pair 11 (Corn_OC) to be used to expand HCoV-HKU1/OC43, as two shown in sequence in sequence table 21 and sequence 22
Bar single strand dna forms；The primer pair 12 (Corn_NL) is used to expand HCoV-229E/NL63, by sequence in sequence table
Two single strand dnas composition shown in row 23 and sequence 24；The primer pair 13 (hMPV_TY) is used to expand the inclined tuberculosis of people
Poison, it is made up of two single strand dnas shown in sequence in sequence table 25 and sequence 26；The primer pair 14 (Entv_TY) is used
In amplification enterovirus, it is made up of two single strand dnas shown in sequence in sequence table 27 and sequence 28；The primer pair 15
(EV71_VP1) it is used to expand enterovirus EV 71 type, as two single stranded DNAs shown in sequence in sequence table 29 and sequence 30 point
Son composition；The primer pair 16 (CA16_VP1) is used to expand enterovirus CA16 types, by sequence in sequence table 31 and sequence 32
Shown two single strand dnas composition.The internal reference primer pair (GAPD_IC) is used to expand house-keeping gene in human genome
GAPDH mRNA, it is made up of two single strand dnas shown in sequence in sequence table 49 and sequence 50.
The ssDNA probe 1 (InfA_TY_MB) is used for the amplification for monitoring the primer pair 1 in real time, its nucleosides
Acid sequence is sequence 33 in sequence table；The ssDNA probe 2 (InfA_H1_MB) is used to monitor the primer pair 2 in real time
Amplification, its nucleotides sequence are classified as sequence 34 in sequence table；The ssDNA probe 3 (InfA_H3_MB) is used to supervise in real time
The amplification of the primer pair 3 is surveyed, its nucleotides sequence is classified as sequence 35 in sequence table；(the InfB_ of ssDNA probe 4
TY_MB) it is used for the amplification for monitoring the primer pair 4 in real time, its nucleotides sequence is classified as sequence 36 in sequence table；It is described single-stranded
DNA probe 5 (RSV_TY_MB) is used for the amplification for monitoring the primer pair 5 in real time, and its nucleotides sequence is classified as sequence in sequence table
Row 37；The ssDNA probe 6 (HRV_TY_MB) is used for the amplification for monitoring the primer pair 6 in real time, its nucleotides sequence
It is classified as sequence 38 in sequence table；The ssDNA probe 7 (ADV_TY_MB) is used for the amplification knot for monitoring the primer pair 7 in real time
Fruit, its nucleotides sequence are classified as sequence 39 in sequence table；The ssDNA probe 8 (PIV1_TY_MB) is used for described in monitoring in real time
The amplification of primer pair 8, its nucleotides sequence are classified as sequence 40 in sequence table；The ssDNA probe 9 (PIV2_TY_MB) is used
In the amplification for monitoring the primer pair 9 in real time, its nucleotides sequence is classified as sequence 41 in sequence table；The ssDNA probe
10 (PIV3_TY_MB) are used for the amplification for monitoring the primer pair 10 in real time, and its nucleotides sequence is classified as sequence in sequence table
42；The ssDNA probe 11 (Corn_OC_MB) is used for the amplification for monitoring the primer pair 11 in real time, its nucleotides sequence
It is classified as sequence 43 in sequence table；The ssDNA probe 12 (Corn_NL_MB) is used for the expansion for monitoring the primer pair 12 in real time
Increase result, its nucleotides sequence is classified as sequence 44 in sequence table；The ssDNA probe 13 (hMPV_TY_MB) is used to monitor in real time
The amplification of the primer pair 13, its nucleotides sequence are classified as sequence 45 in sequence table；(the Entv_ of ssDNA probe 14
TY_MB) it is used for the amplification for monitoring the primer pair 14 in real time, its nucleotides sequence is classified as sequence 46 in sequence table；The list
Ssdna probe 15 (EV71_VP1_MB) is used for the amplification for monitoring the primer pair 15 in real time, and its nucleotides sequence is classified as sequence
Sequence 47 in table；The ssDNA probe 16 (CA16_VP1_MB) is used for the amplification for monitoring the primer pair 16 in real time,
Its nucleotides sequence is classified as sequence 48 in sequence table.The internal reference probe (GAPD_IC_MB) is used to monitor the internal reference in real time
The amplification of primer pair (GAPD_IC), its nucleotides sequence are classified as sequence 51 in sequence table.Every ssDNA probe with
And 5 ' the ends mark of the internal reference probe (GAPD_IC_MB) has FAM, 3 ' ends mark, which has, to be quenched
Go out group TAMRA.
Wherein, the method for primer and probe being embedded into dish-style chip is：Primer, corresponding probe, agarose are mixed,
Be configured to three kinds of compositions final concentration be followed successively by 0.2 μM, 50nM, the mixed solution of 0.1% (weight/mass percentage composition), take 1 μ l should
Solution clicks and enters corresponding dish-style chip reaction chamber, and after being dried in clean super-clean bench, tabletting encapsulation, punching press is made after vacuumizing.
2nd, for the application method for the kit for detecting Respirovirus
1st, reaction system is prepared
15 μ l constant-temperature amplifications buffer solutions (see step 1 1), 10 μ l constant-temperature amplifications enzyme solutions (see step 1 2), 25 μ l are taken to treat
This liquid of test sample is mixed into 50 μ l reaction solutions, after vortex concussion uniformly injection be mounted with primer pair and the 24 of ssDNA probe anti-
Chamber dish-style chip (see step 1 3) is answered, seals Quick spin 30s after membrana oralis.
2nd, isothermal amplification reactions and detection
Dish-style chip is placed in rich Isochip-RT constant-temperature amplifications instrument difficult to understand, sets 41 DEG C to react 1 hour, and complete simultaneously
Real-time fluorescence scans.
3rd, result judges
After reaction terminates, the sample to be tested liquid is determined according to the amplification curve of sample in each reaction chamber as follows
In whether contain corresponding Respirovirus genome：If the testing sample produces S type amplification curves, the sample to be tested
Contain the genome of corresponding Respirovirus in liquid；Conversely, then corresponding Respirovirus is not contained in the sample to be tested liquid
Genome.
Embodiment 2, the analysis of the sensitivity and specificity of kit for detecting Respirovirus
First, the preparation of reference material RNA nucleic acid
1st, structure includes the plasmid of viral targets gene
(1) plasmid containing influenza A virus NP target genes
By 1~1541 section (the influenza A virus NP target gene sequences of influenza A virus NP target gene sequences
Genbank Sequence ID:Gb | KM366530.1 |, Update Date：2014-9-27) it is inserted into pUC19 carriers
Between the multiple cloning sites EcoR V of (day is with biochemical corp's product), recombinant plasmid pUC19-InfA is obtained.
(2) plasmid containing influenza A virus H1 hypotype target genes
By 1~1698 section (the influenza A virus H1 hypotype targets of influenza A virus H1 hypotype target gene sequences
The Genbank Sequence ID of gene order:Gb | GQ475727.1 |, Update Date：2009-8-19) it is inserted into
Between pUC19 carriers (day is with biochemical corp's product) EcoR V, recombinant plasmid pUC19-InfA_H1 is obtained.
(3) plasmid containing influenza A virus H3 hypotype target genes
By 1~1701 section (the influenza A virus H3 hypotype targets of influenza A virus H3 hypotype target gene sequences
The Genbank Sequence ID of gene order:Gb | KJ567658.1 |, Update Date：2014-4-6) it is inserted into
Between the multiple cloning sites EcoR V of pUC19 carriers (day is with biochemical corp's product), recombinant plasmid pUC19-InfA_H3 is obtained.
(4) plasmid containing influenza B virus MP target genes
By 2~1100 sections (the influenza B virus MP target gene sequences of influenza B virus MP target gene sequences
Genbank Sequence ID:Gb | EU305617.1 |, Update Date：2007-12-5) it is inserted into pUC19 carriers
Between the multiple cloning sites EcoR V of (day is with biochemical corp's product), recombinant plasmid pUC19-InfB is obtained.
(5) plasmid containing Respiratory Syncytial Virus(RSV) target gene
By 5680~7390 sections (the Respiratory Syncytial Virus(RSV) target gene sequence of Respiratory Syncytial Virus(RSV) target gene sequence
The Genbank Sequence ID of row:Gb | KJ627648.1 |, Update Date：2014-4-16) it is inserted into pUC19 carriers
Between the multiple cloning sites EcoR V of (day is with biochemical corp's product), recombinant plasmid pUC19-RSV is obtained.
(6) plasmid containing rhinovirus target gene
By 1~1175 section (No. Genbank of rhinovirus target gene sequence of rhinovirus target gene sequence
Sequence ID:Gb | M16248.1 |, Update Date：PUC19 carriers 2003-2-7) are inserted into (to produce with biochemical corp in day
Product) multiple cloning sites EcoR V between, obtain recombinant plasmid pUC19-HRV.
(7) plasmid containing adenovirus target gene
By 20035-22649 sections (No. Genbank of adenovirus target gene sequence of adenovirus target gene sequence
Sequence ID:Gb | KF268314.1 |, Update Date：2013-9-12) being inserted into pUC19 carriers, (day is with biochemical corp
Product) multiple cloning sites EcoR V between, obtain recombinant plasmid pUC19-ADV.
(8) plasmid containing parainfluenza virus I type target genes
By 464~1296 sections (the parainfluenza virus I type target gene sequences of parainfluenza virus I type target gene sequences
Genbank Sequence ID:Gb | JF416791.1 |, Update Date：2014-12-31) it is inserted into pUC19 carriers
Between the multiple cloning sites EcoR V of (day is with biochemical corp's product).Obtain recombinant plasmid pUC19-PIV1.
(9) plasmid containing parainfluenza virus II type target genes
By 2568~3629 sections (the parainfluenza virus II type target gene sequences of parainfluenza virus II type target gene sequences
The Genbank Sequence ID of row:Gb | DQ072586.1 |, Update Date：2008-9-4) it is inserted into pUC19 carriers
Between the multiple cloning sites EcoR V of (day is with biochemical corp's product).Obtain recombinant plasmid pUC19-PIV2.
(10) plasmid containing parainfluenza virus type III target gene
By 211~1190 sections (the parainfluenza virus type III target gene of parainfluenza virus type III target gene sequence
The Genbank Sequence ID of sequence:Dbj | AB623488.1 |, Update Date：2014-7-26) it is inserted into pUC19
Between the multiple cloning sites EcoR V of carrier (day is with biochemical corp's product).Obtain recombinant plasmid pUC19-PIV3.
(11) plasmid of HCoV-HKU1/OC43 target genes is contained
By 20438~21549 sections (HCoV-HKU1/OC43 targets of HCoV-HKU1/OC43 target gene sequences
Mark the Genbank Sequence ID of gene order:Gb | KF686344.1 |, Update Date：2014-9-26) it is inserted into
Between the multiple cloning sites EcoR V of pUC19 carriers (day is with biochemical corp's product), recombinant plasmid pUC19-Corn_OC is obtained.
(12) plasmid of HCoV-229E/NL63 target genes is contained
By 15715~17379 sections (HCoV-229E/NL63 targets of HCoV-229E/NL63 target gene sequences
Mark the Genbank Sequence ID of gene order:Gb | JQ765566.1 |, Update Date：2014-9-26) it is inserted into
Between the multiple cloning sites EcoR V of pUC19 carriers (day is with biochemical corp's product), recombinant plasmid pUC19-Corn_NL is obtained.
(13) plasmid containing human metapneumovirus target gene
By 3513~4544 sections of human metapneumovirus target gene sequence (human metapneumovirus target gene sequence
Genbank Sequence ID:Gb | KC562240.1 |, Update Date：2013-4-21) be inserted into pUC19 carriers (my god
With biochemical corp's product) multiple cloning sites EcoR V between, obtain recombinant plasmid pUC19-hMPV.
(14) plasmid containing enterovirus target gene
By 5~1705 sections of enterovirus Entv target gene sequences (enterovirus Entv target gene sequences
Genbank Sequence ID:Gb | KC755230.1 |, Update Date：2013-12-17) be inserted into pUC19 carriers (my god
With biochemical corp's product) multiple cloning sites EcoR V between, obtain recombinant plasmid pUC19-Entv.
(15) plasmid containing enterovirus EV 71 target gene
By 4~1695 sections of enterovirus EV 71 target gene sequence (enterovirus EV 71 target gene sequence
Genbank Sequence ID:Gb | HM245928.1 |, Update Date：2011-9-27) be inserted into pUC19 carriers (my god
With biochemical corp's product) multiple cloning sites EcoR V between, obtain recombinant plasmid pUC19-EV71.
(16) plasmid containing enterovirus CA16 target genes
By 1392~2564 sections of enterovirus CA16 target gene sequences (enterovirus CA16 target gene sequences
Genbank Sequence ID:Gb | AY790926.1 |, Update Date：2005-8-9) be inserted into pUC19 carriers (day with
Biochemical corp's product) multiple cloning sites EcoR V between, obtain recombinant plasmid pUC19-CA16.
2nd, the preparation of reference material RNA nucleic acid
For trying recombinant plasmid：16 kinds of recombinant plasmids for containing corresponding viral targets gene that step 1 is built.
(1) digestion：First 37 DEG C of digestion 2h of EcoRI restriction endonucleases are used by each for examination recombinant plasmid.
(2) transcribe：It polymerize by 5 μ l 5 × Transcription Optimized Buffer (Promega), 2U T7RNA
Enzyme, 10mM DTT (Promega), 10U recombinant RNAs enzyme inhibitor (Promega), 2mM rNTP, the μ l of digestion products 5 prepare overall
Product is 50 μ l reaction system, 37 DEG C of transcription 4h after shaking uniformly.
(3) digest：System after the completion of transcription is added to 1 μ l DNA digestive ferments (rDnaseI, 5U/ μ l), concussion centrifuges,
37 DEG C of incubation 20min.
(4) purify：Using the RNA purification kits products of Macherey-Nagel companies, (its catalog number is
740948) transcription product RNA is purified as follows：A) RA1-C is prepared2H5OH mixed liquors：With RA1:C2H5OH=1:1 (volume
Than) ratio prepare., it is necessary to add 600 μ l RA1-C in every 100 μ l transcription product2H5OH mixed liquors, i.e. 300 μ l RA1+
300μl C2H5OH solution, need to calculate the volume for matching somebody with somebody mixed liquor according to the pipe number of transcription product herein.If product is insufficient
100 μ l, then the amount of product is mended to 100 μ l with water and (50 μ l Tiangeng Rnase-free H are added in every pipe product2O)。
B) the ready RA1-C by before2H5OH mixed liquors are assigned in 1.5ml centrifuge tubes, two pipes, often the μ l of pipe 600, and 100 μ l products are turned
Enter into corresponding centrifuge tube, totally 700 μ l, fully shaking centrifuge in pipe.C) prepare two adsorption columns, and carry out corresponding mark
Note, 700 μ l product mixtures are transferred in adsorption column (adsorption column maximum capacity is 700 μ l), 1.2 ten thousand rpm centrifugation 2min, are fallen
Fall down a layer solution.D) 700 μ l RA3 is added in adsorption column, 1min is placed, it is sufficiently dispersed in the bottom of adsorption column
Portion, 1.2 ten thousand rpm centrifugation 2min, outwells lower floor's solution.E) 350 μ l RA3 is added in adsorption column, places 2min, 1.2 ten thousand rpm
2min is centrifuged, outwells lower floor's solution.F) 300 μ l RA3 is added in adsorption column, 2min is placed, 1.2 ten thousand rpm centrifugation 2min, falls
Fall down a layer solution.G) after the lid 3min for opening adsorption column, 1.2 ten thousand rpm skies outwell lower floor's solution, repeat this step from 2min
Once.H) it is empty from end after, adsorption column is transferred in corresponding centrifuge tube, opens adsorption column lid and place 15min, make
Ethanol volatilization is complete, and 60 μ l Rnase-H are added in adsorption column2O (carries) elution in kit, place 2min, and 1.2 ten thousand
Rpm centrifuges 2min.I) centrifugation gained template is sucked back in adsorption column, places 2min, 1.2 ten thousand rpm centrifugation 2min, repeat this step
Twice.J) adsorption column is lost, the template in centrifuge tube is taken out into 2 μ l, determines its concentration with Nano drop, and record its concentration
And its 260/280,260/230 ratio.
2nd, for the sensitivity and specificity analysis for the kit for detecting Respirovirus
1st, prepared by the reference material template of various concentrations
Each RNA templates (the 16 kinds of recombinant plasmids built in corresponding step 1 1) of above-mentioned steps one after purification are calculated
Quantify to 1010Copy/μ l, and gradient dilution, obtain 105Copy/μ l, 104Copy/μ l, 103Copy/μ l, 102Copy/μ l, 10
The template dilution of the various concentrations such as copy/μ l.
2nd, reaction system is prepared
15 μ l constant-temperature amplifications buffer solutions are taken (see the step 1) of embodiment 1,10 μ l constant-temperature amplifications enzyme solutions (see the step of embodiment 1
2), the template dilution of 25 μ l concentration is mixed into 50 μ l reaction solutions, after vortex concussion uniformly injection be mounted with primer pair and
24 reaction chamber dish-style chips of ssDNA probe (see the step 3) of embodiment 1, seal Quick spin 30s after membrana oralis.
3rd, isothermal amplification reactions and detection
Dish-style chip is placed in rich Isochip-RT constant-temperature amplifications instrument difficult to understand, sets 41 DEG C to react 1 hour, and complete simultaneously
Real-time fluorescence scans.And result is judged according to the method for the step 23 of embodiment 1.
3rd, result
As a result it is as shown in Figure 2：
(1) for recombinant plasmid pUC19-InfA RNA sample, the 1# reaction chambers pair of 24 reaction chamber dish-style chips
105Copy/μ l, 104Copy/μ l, 103Copy/μ l, 102The testing result of copy/μ l, 10 copies/μ l templates has obvious S
Type amplification curve, it is shown as positive；And 2#-16# reaction chambers are shown as negative without amplification curve.
(2) for recombinant plasmid pUC19-InfA_H1 RNA sample, the 2# reaction chambers of 24 reaction chamber dish-style chips
To 105Copy/μ l, 104Copy/μ l, 103Copy/μ l, 102The testing result of copy/μ l, 10 copies/μ l templates has substantially
S type amplification curves, be shown as positive；And 1#, 3#-16# reaction chamber are shown as negative without amplification curve.
(3) for recombinant plasmid pUC19-InfA_H3 RNA sample, the 3# reaction chambers of 24 reaction chamber dish-style chips
To 105Copy/μ l, 104Copy/μ l, 103Copy/μ l, 102The testing result of copy/μ l, 10 copies/μ l templates has substantially
S type amplification curves, be shown as positive；And 1#, 2# and 4-16# reaction chamber are shown as negative without amplification curve.
(4) for recombinant plasmid pUC19-InfB RNA sample, the 4# reaction chambers pair of 24 reaction chamber dish-style chips
105Copy/μ l, 104Copy/μ l, 103Copy/μ l, 102The testing result of copy/μ l, 10 copies/μ l templates has obvious S
Type amplification curve, it is shown as positive；And 1#-3# and 5#-16# reaction chambers are shown as negative without amplification curve.
(5) for recombinant plasmid pUC19-RSV RNA sample, the 5# reaction chambers of 24 reaction chamber dish-style chips are to 105
Copy/μ l, 104Copy/μ l, 103Copy/μ l, 102The testing result of copy/μ l, 10 copies/μ l templates has obvious S types
Amplification curve, it is shown as positive；And 1#-4# and 6#-16# reaction chambers are shown as negative without amplification curve.
(6) for recombinant plasmid pUC19-HRV RNA sample, the 6# reaction chambers of 24 reaction chamber dish-style chips are to 105
Copy/μ l, 104Copy/μ l, 103Copy/μ l, 102The testing result of copy/μ l, 10 copies/μ l templates has obvious S types
Amplification curve, it is shown as positive；And 1#-5# and 7#-16# reaction chambers are shown as negative without amplification curve.
(7) for recombinant plasmid pUC19-ADV RNA sample, the 7# reaction chambers of 24 reaction chamber dish-style chips are to 105
Copy/μ l, 104Copy/μ l, 103Copy/μ l, 102The testing result of copy/μ l, 10 copies/μ l templates has obvious S types
Amplification curve, it is shown as positive；And 1#-6# and 8#-16# reaction chambers are shown as negative without amplification curve.
(8) for recombinant plasmid pUC19-PIV1 RNA sample, the 8# reaction chambers pair of 24 reaction chamber dish-style chips
105Copy/μ l, 104Copy/μ l, 103Copy/μ l, 102The testing result of copy/μ l, 10 copies/μ l templates has obvious S
Type amplification curve, it is shown as positive；And 1#-7# and 9#-16# reaction chambers are shown as negative without amplification curve.
(9) for recombinant plasmid pUC19-PIV2 RNA sample, the 9# reaction chambers pair of 24 reaction chamber dish-style chips
105Copy/μ l, 104Copy/μ l, 103Copy/μ l, 102The testing result of copy/μ l, 10 copies/μ l templates has obvious S
Type amplification curve, it is shown as positive；And 1#-8# and 10#-16# reaction chambers are shown as negative without amplification curve.
(10) for recombinant plasmid pUC19-PIV3 RNA sample, the 10# reaction chambers pair of 24 reaction chamber dish-style chips
105Copy/μ l, 104Copy/μ l, 103Copy/μ l, 102The testing result of copy/μ l, 10 copies/μ l templates has obvious S
Type amplification curve, it is shown as positive；And 1#-9# and 11#-16# reaction chambers are shown as negative without amplification curve.
(11) for recombinant plasmid pUC19-Corn_OC RNA sample, the 11# reactions of 24 reaction chamber dish-style chips
Chamber is to 105Copy/μ l, 104Copy/μ l, 103Copy/μ l, 102The testing result of copy/μ l, 10 copies/μ l templates has bright
Aobvious S type amplification curves, it is shown as positive；And 1#-10# and 12#-16# reaction chambers are shown as negative without amplification curve.
(12) for recombinant plasmid pUC19-Corn_NL RNA sample, the 12# reactions of 24 reaction chamber dish-style chips
Chamber is to 105Copy/μ l, 104Copy/μ l, 103Copy/μ l, 102The testing result of copy/μ l, 10 copies/μ l templates has bright
Aobvious S type amplification curves, it is shown as positive；And 1#-11# and 13#-16# reaction chambers are shown as negative without amplification curve.
(13) for recombinant plasmid pUC19-hMPV RNA sample, the 13# reaction chambers pair of 24 reaction chamber dish-style chips
105Copy/μ l, 104Copy/μ l, 103Copy/μ l, 102The testing result of copy/μ l, 10 copies/μ l templates has obvious S
Type amplification curve, it is shown as positive；And 1#-12# and 14#-16# reaction chambers are shown as negative without amplification curve.
(14) for recombinant plasmid pUC19-Entv RNA sample, the 14# reaction chambers pair of 24 reaction chamber dish-style chips
105Copy/μ l, 104Copy/μ l, 103Copy/μ l, 102The testing result of copy/μ l, 10 copies/μ l templates has obvious S
Type amplification curve, it is shown as positive；And 1#-13# and 15#-16# reaction chambers are shown as negative without amplification curve.
(15) for recombinant plasmid pUC19-EV71 RNA sample, the 15# reaction chambers pair of 24 reaction chamber dish-style chips
105Copy/μ l, 104Copy/μ l, 103Copy/μ l, 102The testing result of copy/μ l, 10 copies/μ l templates has obvious S
Type amplification curve, it is shown as positive；And 1#-14# and 16# reaction chambers are shown as negative without amplification curve.
(16) for recombinant plasmid pUC19-CA16 RNA sample, the 16# reaction chambers pair of 24 reaction chamber dish-style chips
105Copy/μ l, 104Copy/μ l, 103Copy/μ l, 102The testing result of copy/μ l, 10 copies/μ l templates has obvious S
Type amplification curve, it is shown as positive；And 1#-15# reaction chambers are shown as negative without amplification curve.
In addition, for the RNA sample of 16 kinds of all recombinant plasmids, the 17# reaction chambers of 24 reaction chamber dish-style chips
(internal reference) and 18# reaction chambers (negative control) are without amplification curve.17# reaction chambers (internal reference) why without amplification curve be
Because sample to be tested is the RNA nucleic acid of 16 kinds of recombinant plasmids, the RNA of house-keeping gene GAPDH in human genome is not contained.
It these results suggest that the kit has higher amplification sensitivity and specific amplification.
The detection of embodiment 3, actual clinical sample
First, clinical sample type
The present embodiment use clinical sample from Shenzhen San Yuan in collection Nasopharyngeal swabs sample (in line with this picker from
The principle of hope), adopt swab and deposit in 3mL physiological saline, totally 560.
2nd, in clinical sample viral nucleic acid extraction
Clinical sample viral nucleic acid extraction kit used is QIAamp Viral RNA Mini Kit (Qiagen),
Extracted as follows：
(1) take and the μ l of physiological saline 140 of clinical sample swab are deposited in step 1 in 1.5ml centrifuge tubes；
(2) Buffer AVL (the i.e. 5.6 μ l Carrier RNA mixing that 560 μ l contain Carrier RNA mixed liquors is added
The μ l Buffer of liquid+560 AVL are in centrifuge tube, slight whirlpool 15s；
(3) after the completion of brief centrifugation, (15-25 DEG C) placement 10-20min of room temperature, to ensure Buffer AVL in centrifuge tube
In have abundance time cracked；
(4) 560 μ l ethanol (96-100%, volumn concentration) are added in centrifuge tube, whirlpool mixes 15s, centrifugation；
(5) 630 μ l sample solutions are taken in adsorption column, 2min is placed, it is fully contacted with adsorption column, 8000rpm, from
Heart 1min, change clean collecting pipe；
(6) if sample solution is more than 630 μ l, repeatedly previous step；
(7) 500 μ l AW1 washing lotions are added in adsorption column, 2min is placed, it is fully contacted with adsorption column, 8000rpm
1min is centrifuged, changes clean collecting pipe；
(8) 500 μ l AW2 washing lotions are added in adsorption column, 2min is placed, it is fully contacted with adsorption column,
12000rpm centrifuges 3min, changes clean collecting pipe；
(9) 300 μ l AW2 washing lotions are added in adsorption column, 2min is placed, it is fully contacted with adsorption column,
12000rpm centrifuges 3min, changes clean collecting pipe；
(10) adsorption column lid is opened and places 2min, make that its inside and outside pressure is consistent, and 8000rpm skies are from 1min.It is empty from knot
Shu Yihou, repeat this step once.
(11) it is empty from end after, adsorption column is transferred in 1.5ml centrifuge tubes, open adsorption column lid, place 20-30min,
Make ethanol volatilization complete；
(12) 50 μ l Buffer AVE elution nucleic acid, 8000rpm centrifugation 1min, after centrifugation is completed, by under elution are added
Nucleic acid suck back adsorption column, lose adsorption column after centrifuging 1min.
3rd, the detection of actual clinical sample
1st, reaction system is prepared
15 μ l constant-temperature amplifications buffer solutions (see the step 11 of embodiment 1), 10 μ l constant-temperature amplifications enzyme solutions are taken (see the step of embodiment 1
Rapid 1 2), the clinical sample solution that 25 μ l step 2 are extracted to obtain is mixed into 50 μ l reaction solutions, is injected after vortex concussion uniformly
It is mounted with the 24 reaction chamber dish-style chips (see the step 13 of embodiment 1) of primer pair and ssDNA probe, seals rapid after membrana oralis
Centrifuge 30s.
2nd, isothermal amplification reactions and detection
Dish-style chip is placed in rich Isochip-RT constant-temperature amplifications instrument difficult to understand, sets 41 DEG C to react 1 hour, and complete simultaneously
Real-time fluorescence scans.And result is judged according to the method for the step 23 of embodiment 1.
Experiment sets reliability demonstration of the conventional RT-PCR method for kit testing result of the present invention simultaneously.For examining
The specific RT-PCR primer sequence for surveying each Respirovirus is as shown in table 1.
Table 1 is used for the specific RT-PCR primer sequence for detecting 16 kinds of Respiroviruses
| Index name | RT-PCR sense primers (5 ' -3 ') | RT-PCR anti-sense primers (5 ' -3 ') |
| Influenza A virus | CTTGTCTTTAGCCAYTCCATGAGAGC | CTAACCGAGGTCGAAACGTAYGTTCT |
| Influenza A virus H1 hypotypes | CATCTAYCATTCCAGTCCACCCCCCT | ACCCCAGAAATAGCHAAAAGACCCAA |
| Influenza A virus H3 hypotypes | GTGCTITTAATCAAAAGTATGTCTCCCG | CACAGTITCTACCAAAAGAAGCCAAC |
| Influenza B virus | TTCAGGTACATGACCATGAGACARTA | TYGGTGGGAAAGAATTTGACCTAGAC |
| Parainfluenza virus I types | GTAGTCTCATTCACAGTGGGYAAGGA | ATCTCATTATTACCYGGACCAAGTCT |
| Parainfluenza virus II types | TACAAGACACAACCTCCTGGTATAGCA | GGACGCCTAAATATGGACCTCTCCT |
| Parainfluenza virus type III | TCTCTCTGTGTTTTCCCVGGACACCC | GACTTAAATCCYAGGATCTCTCATAC |
| HCoV-HKU1/OC43 | AGGCTGTATGATGAATGTTGCTAAG | CCTGCACCTAAATGYAAAACWCGCATAT |
| HCoV-229E/NL63 | CAATACAGGGTCCTCCTGGTAGTGG | CACTACCAGGAGGACCCTGTATTGT |
| Respiratory Syncytial Virus(RSV) | ACTGATCCTGCATTITCACARTACCA | AGGTGTAACIACACCTITAAGCACTTA |
| Rhinovirus | TCYAGCCTGCGTGGCTGCCTGC | GAAACACGGACACCCAAAGTAGTYGGT |
| Adenovirus | TGYAACATGACCAARGACTGGTTC | GAAGGGTGGRCTCRTCCATGGGCTC |
| Human metapneumovirus | GCAACATTGAAYTGATCYTCAGGAA | ACCCRTGCAAAGTYAGCACAGGAAG |
| Enterovirus | CCCCTGAATGCGGCTAATCCYAAC | CCAATCCAATAGCTATATGGYAACAA |
| Enterovirus EV 71 type | AACACCCGTAYGTGCTTGATGCTGG | TAGGGATTACTGGCGTCGCTCCTTG |
| Enterovirus CA16 types | CACCCCCATGCARCGCTTGTGYTTT | TRGACTGCCACGGRCCATCTCTTCC |
4th, result
The testing result of kit of the present invention is shown, for each Respirovirus, sun is detected using RT-PCR method
Property sample example using kit of the present invention detection show positive findings.In addition, for a few sample example, using RT-
The detection of PCR methods is negative, but uses kit testing result of the present invention as the positive.Adopted again by the way that these sample examples are carried out with the later stage
Sample RT-PCR is detected, it is found that it is positive these sample examples are detected as corresponding Respirovirus really.It is indicated above, kit of the present invention
RT-PCR methods are higher than to the recall rate of corresponding Respirovirus, and its testing result is accurately and reliably.Kit and RT- of the present invention
The statistical result of PCR methods detection common respiratory tract virus clinical sample is referring to table 2.
The statistical result of 2 kit of the present invention of table and RT-PCR methods detection common respiratory tract virus clinical sample
Claims (3)
1. the kit for detecting Respirovirus, contains complete single stranded DNA；The complete single stranded DNA, by probe groups and draw
Thing is to a group composition；
The primer pair group, it is made up of following 16 primer pairs：
The primer pair 1 being made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2；
The primer pair 2 being made up of two single strand dnas shown in sequence in sequence table 3 and sequence 4；
The primer pair 3 being made up of two single strand dnas shown in sequence in sequence table 5 and sequence 6；
The primer pair 4 being made up of two single strand dnas shown in sequence in sequence table 7 and sequence 8；
The primer pair 5 being made up of two single strand dnas shown in sequence in sequence table 9 and sequence 10；
The primer pair 6 being made up of two single strand dnas shown in sequence in sequence table 11 and sequence 12；
The primer pair 7 being made up of two single strand dnas shown in sequence in sequence table 13 and sequence 14；
The primer pair 8 being made up of two single strand dnas shown in sequence in sequence table 15 and sequence 16；
The primer pair 9 being made up of two single strand dnas shown in sequence in sequence table 17 and sequence 18；
The primer pair 10 being made up of two single strand dnas shown in sequence in sequence table 19 and sequence 20；
The primer pair 11 being made up of two single strand dnas shown in sequence in sequence table 21 and sequence 22；
The primer pair 12 being made up of two single strand dnas shown in sequence in sequence table 23 and sequence 24；
The primer pair 13 being made up of two single strand dnas shown in sequence in sequence table 25 and sequence 26；
The primer pair 14 being made up of two single strand dnas shown in sequence in sequence table 27 and sequence 28；
The primer pair 15 being made up of two single strand dnas shown in sequence in sequence table 29 and sequence 30；
The primer pair 16 being made up of two single strand dnas shown in sequence in sequence table 31 and sequence 32；
The probe groups are made up of following 16 ssDNA probes：SsDNA probe 1 in sequence table shown in sequence 33；Sequence
SsDNA probe 2 in table shown in sequence 34；SsDNA probe 3 in sequence table shown in sequence 35；Sequence 36 in sequence table
Shown ssDNA probe 4；SsDNA probe 5 in sequence table shown in sequence 37；It is single-stranded shown in sequence 38 in sequence table
DNA probe 6；SsDNA probe 7 in sequence table shown in sequence 39；SsDNA probe 8 in sequence table shown in sequence 40；Sequence
SsDNA probe 9 in list shown in sequence 41；SsDNA probe 10 in sequence table shown in sequence 42；Sequence in sequence table
SsDNA probe 11 shown in 43；SsDNA probe 12 in sequence table shown in sequence 44；In sequence table shown in sequence 45
SsDNA probe 13；SsDNA probe 14 in sequence table shown in sequence 46；Single stranded DNA in sequence table shown in sequence 47 is visited
Pin 15；SsDNA probe 16 in sequence table shown in sequence 48；
5 ' the ends mark of every ssDNA probe in the probe groups has FAM, and 3 ' ends mark has
Optical quenching group TAMRA；
The kit also contains internal reference primer pair and internal reference probe；The internal reference primer pair is by sequence in sequence table 49
With two single strand dnas composition shown in sequence 50；The internal reference probe is the single stranded DNA shown in sequence 51 in sequence table
Probe；5 ' ends of the internal reference probe are marked with fluorescent reporter group FAM, and 3 ' ends are marked with fluorescent quenching group TAMRA；
Also contain dish-style chip in the kit；
The 1# of the dish-style chip deposits dry following (1)-(18) to 18# reaction chambers respectively：
(1) primer pair 1 and the ssDNA probe 1；
(2) primer pair 2 and the ssDNA probe 2；
(3) primer pair 3 and the ssDNA probe 3；
(4) primer pair 4 and the ssDNA probe 4；
(5) primer pair 5 and the ssDNA probe 5；
(6) primer pair 6 and the ssDNA probe 6；
(7) primer pair 7 and the ssDNA probe 7；
(8) primer pair 8 and the ssDNA probe 8；
(9) primer pair 9 and the ssDNA probe 9；
(10) primer pair 10 and the ssDNA probe 10；
(11) primer pair 11 and the ssDNA probe 11；
(12) primer pair 12 and the ssDNA probe 12；
(13) primer pair 13 and the ssDNA probe 13；
(14) primer pair 14 and the ssDNA probe 14；
(15) primer pair 15 and the ssDNA probe 15；
(16) primer pair 16 and the ssDNA probe 16；
(17) the internal reference primer pair and the internal reference probe；
(18) negative controls；
The dish-style chip is 24 reaction chamber dish-style chips.
2. kit according to claim 1, it is characterised in that：In the kit also containing constant-temperature amplification buffer solution and
Constant-temperature amplification enzyme solutions；
The solvent of the constant-temperature amplification buffer solution is water, and solute and concentration are as follows：200mM pH 8.0 Tris-HCL, 50mM
DTT, 10mM dNTP, 10mM rNTP, 80mM MgCl2, 450mM KCl, 15% volumn concentration DMSO, 1M sorbierite,
20mM tetramethyl ammonium chlorides；
The solvent of the constant-temperature amplification enzyme solutions is water, and solute and concentration are as follows：AMV reverse transcriptase 1U/ μ l, T7 RNA polymerases
5U/ μ l, ribonuclease H 0.5U/ μ l, the μ g/ μ l of pyrophosphatase 0.5U/ μ l, RNase inhibitor 5U/ μ l, BSA 0.5.
3. kit according to claim 1 or 2, it is characterised in that：The Respirovirus is at least one in following
Kind：Influenza A virus, influenza A virus H1 hypotypes, influenza A virus H3 hypotypes, influenza B virus, respiratory syncystial
Virus, rhinovirus, adenovirus, parainfluenza virus I types, parainfluenza virus II types, parainfluenza virus type III, HCoV-HKU1/
OC43, HCoV-229E/NL63, human metapneumovirus, enterovirus, enterovirus EV 71 type and enterovirus CA16 types.
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| CN105112559B (en) * | 2015-08-03 | 2017-12-08 | 博奥生物集团有限公司 | A kind of kit and its application for being used to detect coronavirus |
| CN106521035B (en) * | 2016-12-13 | 2019-12-31 | 湖南圣湘生物科技有限公司 | Gene chip detection kit and method for detecting 16 respiratory pathogens |
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| 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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| CN110468238A (en) * | 2019-09-11 | 2019-11-19 | 深圳市芯思微生物科技有限公司 | A kind of primed probe group, kit and the application of constant-temperature amplification detection A type and influenza B virus |
| CN110724764A (en) * | 2019-10-22 | 2020-01-24 | 中国医学科学院病原生物学研究所 | Fluorescent quantitative PCR detection method for human coronavirus and respiratory syncytial virus and application thereof |
| CN111041129B (en) * | 2020-03-13 | 2020-06-05 | 博奥生物集团有限公司 | Primer-probe combination for detecting 6 respiratory viruses, kit and application |
| CN111270020B (en) * | 2020-04-07 | 2023-04-07 | 杭州萧山微岩医学检验实验室有限公司 | Nucleic acid detection reagent and application thereof in novel coronavirus detection |
| CN116287434B (en) * | 2022-10-21 | 2024-03-26 | 深圳康美生物科技股份有限公司 | Respiratory tract syndrome pathogen nucleic acid detection kit |
| CN116606958A (en) * | 2023-03-06 | 2023-08-18 | 广州科方生物技术股份有限公司 | Primer probe composition for detecting parainfluenza 1/2/3, kit and use method |
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