CN108034764A - Multiplex PCR detection Coxsackie virus, enterovirns type 71 and enterovirus universal primed probe group - Google Patents
Multiplex PCR detection Coxsackie virus, enterovirns type 71 and enterovirus universal primed probe group Download PDFInfo
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Abstract
A kind of this disclosure relates to 6 types of multiplex PCR detection Coxsackie virus A/A10 types/A16 types/enterovirns type 71/enterovirus universal primed probe group.The disclosure additionally provides a kind of kit of 6 types of multiple fluorescence quantitative PCR detection Coxsackie virus A/A10 types/A16 types/enterovirns type 71/enterovirus universal, and the kit includes the primed probe group described in the disclosure.The Pass through above-mentioned technical proposal disclosure provides complete solution for the rapid screening of 6 type of Coxsackie virus A in food, A10 types, A16 types, enterovirns type 71 and enterovirus universal, the emergency disposal and integrated prevention and control capacity of hand-foot-and-mouth disease can further be lifted, the guarantee that provides the necessary technical is propagated on a large scale in crowd to reduce hand-foot-and-mouth disease, and then eliminates social influence and economic loss caused by outbreaks of infectious diseases.
Description
Technical field
The present invention relates to biological technical field, and in particular, to 6 types of multiplex PCR detection detection Coxsackie virus A/A10
Type/A16 types/enterovirns type 71/enterovirus universal primed probe group.
Background technology
The detection method of hand-foot-and-mouth disease correlated virus includes virus purification culture, Serological testing and viral nucleic acid detection three
A aspect.First two method is cumbersome, and qualification cycle is grown, and antiserum is easily produced and intersected instead with other Human enterovirus virus
Should.The method of molecular biology method identification Human enterovirus virus's parting is employed more and more, at present existing brothers mouthful
The external diagnosis reagent case of disease includes real time fluorescent PCR method, isothermal duplication PCR method, liquid-phase chip technology etc..With fluorescence mark
Real-time fluorescence PCR technology based on note probe is most widely used in brothers mouthful clinical diagnosis at present, can be quickly and accurately
Sample to be tested is qualitatively detected.According to detection cause of disease number, substance or multiple real time fluorescence PCR skills are further divided into
Art, reaction system include one or more pairs of Specific PCR primers and probe.
The Testing and appraisal technology for detection target of existing hand-foot-and-mouth disease original detection kit is single or just for common Ke's Sa
The joint inspection of strange virus A 16-type, enterovirns type 71, it is difficult to meet the multifarious epidemiology development need of hand-foot-and-mouth disease Viral typing
Ask.There is presently no used for 6 type of Coxsackie virus A, A10 types, A16 types, enterovirns type 71 and enterovirus universal
Multiple real time fluorescence PCR identifies the report of Viral typing at the same time.
The content of the invention
A kind of the problem of being detected at the same time with identification present invention aim to address various serotype enterovirus, there is provided Ke's Sa
The fluorescence quantification PCR primer probe groups of strange virus A6 types/A10 types/A16 types/enterovirns type 71/enterovirus universal and examination
Agent box, while enterovirus disease and 4 kinds of enterovirus serotypes of precise Identification are detected, establish one kind and put down based on quantitative fluorescent PCR
The multi-PCR detection method of platform.
To achieve the above objectives, the present invention provides 6 types of multiplex PCR detection Coxsackie virus A/A10 types/A16 types/enteron aisle
Viral 71 types/enterovirus universal primed probe group, wherein, including component A and component B,
Wherein, the component A includes：
The 6 type sense primer of Coxsackie virus A of nucleotide sequence shown in SEQ ID NO.1,
The 6 type anti-sense primer of Coxsackie virus A of nucleotide sequence shown in SEQ ID NO.2,
6 type probe of Coxsackie virus A with nucleotide sequence shown in SEQ ID NO.3,6 type of Coxsackie virus A
5 ' ends of probe are marked with CY5 luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ3；
The 10 type sense primer of Coxsackie virus A of nucleotide sequence shown in SEQ ID NO.4,
The 10 type anti-sense primer of Coxsackie virus A of nucleotide sequence shown in SEQ ID NO.5,
10 type probe of Coxsackie virus A with nucleotide sequence shown in SEQ ID NO.6, the Coxsackie virus A 10
5 ' ends of type probe are marked with VIC luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ1；
The coxsackie virus A 16-type sense primer of nucleotide sequence shown in SEQ ID NO.7,
The coxsackie virus A 16-type anti-sense primer of nucleotide sequence shown in SEQ ID NO.8,
Coxsackie virus A 16-type probe with nucleotide sequence shown in SEQ ID NO.9, the coxsackie virus A 16
5 ' ends of type probe are marked with FAM luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ1；
The component B includes：
The enterovirns type 71 sense primer of nucleotide sequence shown in SEQ ID NO.10,
The enterovirns type 71 anti-sense primer of nucleotide sequence shown in SEQ ID NO.11,
Enterovirns type 71 probe with nucleotide sequence shown in SEQ ID NO.12, the enterovirns type 71 probe
5 ' ends be marked with VIC luminophores, 3 ' ends are marked with fluorescent quenching group BHQ1；
The enterovirus universal sense primer of nucleotide sequence shown in SEQ ID NO.13,
The enterovirus universal anti-sense primer of nucleotide sequence shown in SEQ ID NO.14,
Enterovirus universal probe with nucleotide sequence shown in SEQ ID NO.15, the enterovirus universal
5 ' ends of probe are marked with FAM luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ1.
Second aspect of the disclosure provides a kind of 6 types of multiple fluorescence quantitative PCR detection Coxsackie virus A/A10 types/A16
The detection method of type/enterovirns type 71/enterovirus universal, wherein, the detection method includes the following steps：
(1) total nucleic acid of sample to be tested is extracted；
(2) using the total nucleic acid as pcr template, using the primed probe group described in disclosure the first aspect to described
Pcr template carries out multiple fluorescence quantitative PCR amplification；
Wherein, the multiple fluorescence quantitative PCR carries out in two systems at the same time,
In first system, the first amplification is carried out to the pcr template using the component A,
In second system, the second amplification is carried out to the pcr template using the component B；
(3) FAM, VIC, CY5 and ROX fluorescence channel signal in first system are collected respectively, are collected in second system
VIC, FAM and ROX fluorescence channel signal.
3rd aspect of the disclosure provides a kind of 6 types of multiple fluorescence quantitative PCR detection Coxsackie virus A/A10 types/A16
The kit of type/enterovirns type 71/enterovirus universal, the kit include drawing described in disclosure the first aspect
Thing probe groups.
6 type of Coxsackie virus A, A10 types, A16 types, enterovirns type 71 and the enterovirus universal that the present invention establishes are more
Weight real-time fluorescence PCR detection method, can quickly realize 6 type of Coxsackie virus A, A10 types, A16 types, Enterovirus 71 in sample
The examination and discriminating of type and enterovirus universal, the troublesome operation for the methods of avoiding serology, cause of disease culture, reach as follows
Detection result：
(1) Multiple detection
The detection method that the present invention is established can be managed by A, B pipe simultaneous reactions, 6 type of examination Coxsackie virus A, A10
Type, A16 types, enterovirns type 71 and enterovirus universal, fast and convenient acquisition hand-foot-and-mouth disease related diseases original seed class, during saving
Between, manpower and reagent cost.
(2) high sensitivity
The detection method that the present invention is established detects while can realizing 6 genes, each target base in reaction system
The detection sensitivity of cause can reach 102PFU/ml is suitable with substance real time fluorescent PCR detection method susceptibility.
(3) specificity is high
The detection method specificity that the present invention is established is mainly reflected in the specificity of a whole set of specific primer probe, institute
There is primer all to compare analysis by BLAST, the conservative and specificity with height, not only detecting target in the present invention can
Mutually distinguish, and can also virus close with other kinds, that living environment is identical differentiate, this include influenza virus, rubella
Virus, measles virus, Respiratory Syncytial Virus(RSV), mumps virus, parainfluenza virus, B races streptococcus, adenovirus, rotavirus,
Varicellazoster virus etc., it was demonstrated that detection method has the specificity of height, can accurately distinguish non-detection target.
(4) false negative result is prevented
Quality Control in the positive added in system, can effectively prompt false negative testing result.
The disclosure is 6 type of Coxsackie virus A, A10 types, A16 types, enterovirns type 71 and enterovirus universal in food
Rapid screening provide complete solution, can further lift the emergency disposal and Synthetical prevention energy of hand-foot-and-mouth disease
Power, the guarantee that provides the necessary technical is propagated to reduce hand-foot-and-mouth disease on a large scale in crowd, and then caused by elimination outbreaks of infectious diseases
Social influence and economic loss.
Brief description of the drawings
Attached drawing is for providing further understanding of the disclosure, and a part for constitution instruction, with following tool
Body embodiment is used to explain the disclosure together, but does not form the limitation to the disclosure.In the accompanying drawings：
Fig. 1 is the testing result of coxsackie virus A 16-type positive clinical throat swab sample A pipes in embodiment 2.
Fig. 2 is the testing result of coxsackie virus A 16-type positive clinical throat swab sample B pipe in embodiment 2.
Fig. 3 is the testing result of 10 type positive clinical throat swab sample A pipes of Coxsackie virus A in embodiment 2.
Fig. 4 is the testing result of 10 type positive clinical throat swab sample B pipe of Coxsackie virus A in embodiment 2.
Fig. 5 is the testing result of 6 type positive clinical throat swab sample A pipes of Coxsackie virus A in embodiment 2.
Fig. 6 is the testing result of 6 type positive clinical throat swab sample B pipe of Coxsackie virus A in embodiment 2.
Fig. 7 is the testing result of enterovirns type 71 positive clinical throat swab sample A pipes in embodiment 2.
Fig. 8 is the testing result of enterovirns type 71 positive clinical throat swab sample B pipe in embodiment 2.
Fig. 9 is the testing result of negative control A pipes in embodiment 2.
Figure 10 is the survey result of negative control inspection B pipes in embodiment 2.
Figure 11 is the testing result of positive control A pipes in embodiment 2.
Figure 12 is the testing result of positive control B pipes in embodiment 2.
Figure 13 is that cause of disease influenza virus, rubella virus, measles virus, Respiratory Syncytial Virus(RSV), the parotid gland are disturbed in embodiment 4
Scorching virus, parainfluenza virus, B races streptococcus, adenovirus, rotavirus, the A pipe testing results of varicellazoster virus.
Figure 14 is that cause of disease influenza virus, rubella virus, measles virus, Respiratory Syncytial Virus(RSV), the parotid gland are disturbed in embodiment 4
Scorching virus, parainfluenza virus, B races streptococcus, adenovirus, rotavirus, the B pipe testing results of varicellazoster virus.
Figure 15 is negative control A pipe testing results in embodiment 4.
Figure 16 is negative control B pipe testing results in embodiment 4.
Figure 17 is positive control A pipe testing results in embodiment 4.
Figure 18 is positive control B pipe testing results in embodiment 4.
Embodiment
The embodiment of the disclosure is described in detail below in conjunction with attached drawing.It should be appreciated that this place is retouched
The embodiment stated is only used for describing and explaining the disclosure, is not limited to the disclosure.
One embodiment of the present invention provides 6 types of multiplex PCR detection Coxsackie virus A/A10 types/A16 types/enteron aisle
Viral 71 types/enterovirus universal primed probe group.
The specific detection gene of primed probe group selection or conserved sequence of the present invention, 6 type of Coxsackie virus A selection clothing
1 gene of glutelin (VP1), 10 type of Coxsackie virus A selection 1 gene of capsid protein (VP1), coxsackie virus A 16-type selection clothing
1 gene of glutelin (VP1), enterovirns type 71 selection 1 gene of capsid protein (VP1), enterovirus universal select 5 ' non-volumes
Code area's gene (5'UTR).
Take into full account that the primed probe of different target gene is common in a reaction system in primed probe group design process
The problem of amplification, therefore, what primed probe to be integrated when designing considers that Tm values uniformity, G/C content are uniformed, while to the greatest extent may be used
It is avoided that situations such as hairpin structure, primer dimer occur, to ensure the probability of later stage different primers probe while amplification.Finally
Obtain a set of special primer probe sequence provided by the invention, including component A and component B.
Wherein, the component A includes：
The 6 type sense primer of Coxsackie virus A of nucleotide sequence shown in SEQ ID NO.1,
The 6 type anti-sense primer of Coxsackie virus A of nucleotide sequence shown in SEQ ID NO.2,
6 type probe of Coxsackie virus A with nucleotide sequence shown in SEQ ID NO.3,6 type of Coxsackie virus A
5 ' ends of probe are marked with CY5 luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ3；
The 10 type sense primer of Coxsackie virus A of nucleotide sequence shown in SEQ ID NO.4,
The 10 type anti-sense primer of Coxsackie virus A of nucleotide sequence shown in SEQ ID NO.5,
10 type probe of Coxsackie virus A with nucleotide sequence shown in SEQ ID NO.6, the Coxsackie virus A 10
5 ' ends of type probe are marked with VIC luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ1；
The coxsackie virus A 16-type sense primer of nucleotide sequence shown in SEQ ID NO.7,
The coxsackie virus A 16-type anti-sense primer of nucleotide sequence shown in SEQ ID NO.8,
Coxsackie virus A 16-type probe with nucleotide sequence shown in SEQ ID NO.9, the coxsackie virus A 16
5 ' ends of type probe are marked with FAM luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ1；
The component B includes：
The enterovirns type 71 sense primer of nucleotide sequence shown in SEQ ID NO.10,
The enterovirns type 71 anti-sense primer of nucleotide sequence shown in SEQ ID NO.11,
Enterovirns type 71 probe with nucleotide sequence shown in SEQ ID NO.12, the enterovirns type 71 probe
5 ' ends be marked with VIC luminophores, 3 ' ends are marked with fluorescent quenching group BHQ1；
The enterovirus universal sense primer of nucleotide sequence shown in SEQ ID NO.13,
The enterovirus universal anti-sense primer of nucleotide sequence shown in SEQ ID NO.14,
Enterovirus universal probe with nucleotide sequence shown in SEQ ID NO.15, the enterovirus universal
5 ' ends of probe are marked with FAM luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ1.
In a kind of preferred embodiment of the disclosure, also respectively containing Quality Control in the positive in the component A and component B
Primed probe,
The nucleotide sequence of Quality Control sense primer is as shown in SEQ ID NO.16 in the positive,
The nucleotide sequence of Quality Control anti-sense primer is as shown in SEQ ID NO.17 in the positive,
The nucleotide sequence of Quality Control probe is as shown in SEQ ID NO.18 in the positive, 5 ' ends of Quality Control probe in the positive
ROX luminophores are marked with, 3 ' ends are marked with fluorescent quenching group BHQ2.
Quality Control primed probe is used to expand plasmid template pET28a in the positive.
In a kind of 6 types of multiple fluorescence quantitative PCR detection Coxsackie virus A/A10 types/A16 types/enterovirus of the present invention
The detection method of 71 types/enterovirus universal, the detection method include the following steps：
(1) total nucleic acid of sample to be tested is extracted；
(2) using the total nucleic acid as pcr template, using the primed probe group described in disclosure the first aspect to described
Pcr template carries out multiple fluorescence quantitative PCR amplification；
Wherein, the multiple fluorescence quantitative PCR carries out in two systems at the same time,
In first system, the first amplification is carried out to the pcr template using the component A,
In second system, the second amplification is carried out to the pcr template using the component B；
(3) FAM, VIC, CY5 and ROX fluorescence channel signal in first system are collected respectively, are collected in second system
VIC, FAM and ROX fluorescence channel signal.
Optionally, the detection method further includes adds Quality Control pET28a plasmid moulds in the positive in two systems respectively
Plate.
In first system, CY5 fluorescence channels are used to detect 6 type of Coxsackie virus A, VIC fluorescence channels for detecting
10 type of Coxsackie virus A, FAM fluorescence channels are used to detect coxsackie virus A 16-type, ROX fluorescence channels for detecting in the positive
Quality Control.
In second system, VIC fluorescence channels are used to detect enterovirns type 71, FAM fluorescence channels for detecting intestines
Road virus is universal, ROX fluorescence channels are used to detect Quality Control in the positive.
In a kind of embodiment of the disclosure, judge by the following method in each fluorescence channel detected material whether be
It is positive：In each fluorescence detection channel：
Each reference substance of kit must reach claimed below, and otherwise experiment is considered as invalid：Quality Control in the positive of negative reference product
There are the amplification of S types, and CtValue is less than 36, and each passage of positive reference product has the amplification of S types, and CtValue is less than 36.
If a) there is the amplification of S types, and CtValue is less than 36, then is determined as that corresponding detected material is positive；
If b) there is the amplification of S types, and CtValue is then determined as uncertain sample, need to extract again less than 40 and more than or equal to 36
Rechecked after nucleic acid；If reinspection still has the amplification of S types, and CtValue be less than 40, then judge corresponding detected material for the positive, otherwise for
It is negative；
If c) without obvious S types amplification curve, but report has CtValue, then be determined as non-specific amplification.
In a kind of specific embodiment of the disclosure, such as following table can be passed through in the case of Quality Control establishment in the positive
1 method is detected the judgement of result.
Table 1
| Result judgement | FAM | VIC | CY5 (or LIZ) | ROX | The universal FAM of enteron aisle |
| CA16 | + | - | - | - | + |
| EV71 | - | + | - | - | + |
| CA6 | - | - | + | - | + |
| CA10 | - | + | - | - | + |
| Other enteroviruses | - | - | - | - | + |
| Enterovirus is negative | - | - | - | - | - |
Optionally, the reaction system of two multiple fluorescence quantitative PCRs of the disclosure includes each component of following content：
Hotstar archaeal dna polymerases containing 1-2U in 20 μ L reaction solutions, the RNase inhibitor of 20-30U, 100-200U
MMLV reverse transcriptases, the Tris-HCl buffer solutions that concentration is 20mM and pH is 8.3, the pET28a, 0.4mM of 0.0003ng/ μ L
DNTP, 4mM MgCl2。
To improve the sensitivity of PCR reactions, present disclose provides specific reaction condition, and there is good effect.
The reaction condition of multiple fluorescence quantitative PCR includes the following steps：
a：45℃ 9-11min；b：95℃ 4-6min；c：95℃ 15-60s；d：50-60 DEG C of 15-60s, c-d circulation 40
A reaction.
Present invention also offers a kind of kit containing foregoing multiple fluorescence quantitative PCR detection primer probe groups.
Preferably, also containing Quality Control plasmid template pET28a in the positive in the kit.
Preferably, the kit further includes 2 × RT-PCR reaction solutions, 10 × primed probe mixture, positive control, the moon
Property control and nuclease-free water.
Preferably, the final concentration of every primer is respectively 0.3-0.6 μM in the kit, and every probe is most
Whole concentration is respectively 0.1-0.3 μM.
Hereinafter, the disclosure, in following embodiments, 6 type of Coxsackie virus A, A10 are further described by embodiment
Type, A16 types, enterovirns type 71 clinical sample are from Chinese People's Liberation Army General Hospital hand-foot-and-mouth disease patient diagnosed and doubtful
The throat swab sample of patient, laboratory is transported to after sample collection.In addition, influenza virus, rubella virus, measles virus, breathing
Road syncytial virus, mumps virus, parainfluenza virus, B races streptococcus, adenovirus, rotavirus, varicellazoster virus
Positive nucleic acid is provided by China Sickness Prevention Control Center Virus Disease Prevention Control Institute.
Embodiment 1
6 types of Coxsackie virus A/A10 types/A16 types/enterovirns type 71/enterovirus universal multiple fluorescence quantitative PCR
The establishment of detection kit：
The composition of primed probe：
A pipe primed probes：The 6 type sense primer of Coxsackie virus A of nucleotide sequence shown in SEQ ID NO.1, SEQ ID
The 6 type anti-sense primer of Coxsackie virus A of nucleotide sequence shown in NO.2, has Ke of nucleotide sequence shown in SEQ ID NO.3
Sa Qi virus A6 type probes, 5 ' ends of the 6 type probe of Coxsackie virus A are marked with CY5 luminophores, and 3 ' ends are marked with fluorescence
Quenching group BHQ3；
The 10 type sense primer of Coxsackie virus A of nucleotide sequence shown in SEQ ID NO.4, core shown in SEQ ID NO.5
The 10 type anti-sense primer of Coxsackie virus A of nucleotide sequence, has the Coxsackie virus of nucleotide sequence shown in SEQ ID NO.6
A10 type probes, 5 ' ends of the 10 type probe of Coxsackie virus A are marked with VIC luminophores, and 3 ' ends are marked with fluorescent quenching base
Group BHQ1；
The coxsackie virus A 16-type sense primer of nucleotide sequence shown in SEQ ID NO.7, core shown in SEQ ID NO.8
The coxsackie virus A 16-type anti-sense primer of nucleotide sequence, has the Coxsackie virus of nucleotide sequence shown in SEQ ID NO.9
A16 type probes, 5 ' ends of the coxsackie virus A 16-type probe are marked with FAM luminophores, and 3 ' ends are marked with fluorescent quenching base
Group BHQ1；
The nucleotide sequence of Quality Control sense primer is as shown in SEQ ID NO.16 in the positive, Quality Control anti-sense primer in the positive
Nucleotide sequence is as shown in SEQ ID NO.17, and the nucleotide sequence of Quality Control probe is as shown in SEQ ID NO.18 in the positive, institute
5 ' the ends for stating Quality Control probe in the positive are marked with ROX luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ2.
B pipe primed probes：The enterovirns type 71 sense primer of nucleotide sequence, SEQ ID shown in SEQ ID NO.10
The enterovirns type 71 anti-sense primer of nucleotide sequence shown in NO.11, has the intestines of nucleotide sequence shown in SEQ ID NO.12
Road 71 type probes of virus, 5 ' ends of the enterovirns type 71 probe are marked with VIC luminophores, and 3 ' ends are marked with fluorescent quenching
Group BHQ1；
The enterovirus universal sense primer of nucleotide sequence shown in SEQ ID NO.13, core shown in SEQ ID NO.14
The enterovirus universal anti-sense primer of nucleotide sequence, the enterovirus with nucleotide sequence shown in SEQ ID NO.15 are general
Type probe, 5 ' ends of the enterovirus universal probe are marked with FAM luminophores, and 3 ' ends are marked with fluorescent quenching group
BHQ1。
The nucleotide sequence of Quality Control sense primer is as shown in SEQ ID NO.16 in the positive, Quality Control anti-sense primer in the positive
Nucleotide sequence is as shown in SEQ ID NO.17, and the nucleotide sequence of Quality Control probe is as shown in SEQ ID NO.18 in the positive, institute
5 ' the ends for stating Quality Control probe in the positive are marked with ROX luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ2.
Kit is by 2 × RT-PCR reaction solutions, 20 × reverse transcriptase, 10 × primed probe mixture, positive control, feminine gender
Control, nuclease-free water are formed, its specific two system components difference is as follows：
2 × RT-PCR reaction solutions (Hotstar archaeal dna polymerases (2U/ μ L), Tris-HCl 100Mm (pH8.3), KCl
100mM, Tween-20 0.2%, 5mM dNTP, 20mM MgCl2), 20 × reverse transcriptase (200U/ μ L)；10 × primed probe
(first system contains the primed probe of component A to mixture, and second system contains the primed probe of component B, comprising in the positive
The concentration of every primer including Quality Control (pET28a plasmid templates) is 0.3 μM, and the concentration of every probe is 0.2 μM), A pipes are
Quality Control primed probe mixed liquor in 6 types of Coxsackie virus A/A10 types/A16 types, the positive, B pipes are enterovirns type 71, and enteron aisle is sick
Poison is universal and internal reference primed probe plasmid template (pET28a 0.0003ng) mixed liquor, A pipes and B pipes are both needed to deposit in palm fibre
Colour tube；
Positive control is the vacation of 6 types of Coxsackie virus A/A10 types/A16 types/enterovirns type 71/enterovirus universal
Viral mixed liquor, every kind of concentration are 104PFU/ml；
Negative control is the physiological saline of autoclave sterilization.
The reaction system of kit detection is 20 μ L, its configuration is as follows：2 × RT-PCR reaction solutions, 10 μ L；20 × reverse transcription
2 μ L of enzyme；10 × primed probe mixture, 2 μ L；5 μ L of RNA templates, 2 μ L of nuclease-free water.
The operation of 2 kit of embodiment and result judge
1st, the extraction of genome
Commodity in use viral genome kit extracts sample rna to be checked as detection sample.
2nd, the preparation of reaction system
With reference to manner of formulation same as Example 1, the PCR pipe of 200 μ L is taken to configure the reaction system of two pipe of A, B, 20 μ L,
Quality Control primed probe mixed liquor in 6 types of Coxsackie virus A/A10 types/A16 types and the positive, B pipes are enterovirus universal/enteron aisle
Quality Control primed probe plasmid template (pET28a 0.0003ng) mixed liquor in viral 71 types and the positive, it configures following 2 × RT-
10 μ L of PCR reaction solution；20 × reverse transcriptase, 1 μ L；10 × primed probe mixture, 2 μ L；5 μ L of RNA templates, 2 μ L of nuclease-free water.
3rd, PCR reacts
PCR pipe is put into fluorescence quantitative PCR instrument, PCR reactions are carried out according to following program：45℃20min；95℃
5min；95 DEG C of 15s, 55 DEG C of 45s, circulate 45 reaction, amplification as shown in figs. 1-12, wherein, IAC represent internal standard control,
CA16 represents coxsackie virus A 16-type, and EU represents the general virus of enteron aisle, and CA10 represents 10 type of Coxsackie virus A, and CA6 represents Ke
Sa Qi virus A6 types, EV71 represent enterovirns type 71.
4th, result judges
1) threshold value is adjusted：Instrument is according to preceding 15 circular response background values, adjust automatically decision threshold.
2) quality control：Quality Control is set up in blank control, positive control and the positive, and it is invalid otherwise to regard experiment.
3) judgement and explanation of each fluorescence detection channel：If a) sample has the amplification of S types, and CT values are less than 36, then judge
Sample is the positive；If b) sample has the amplification of S types, and CT values are less than 40 and more than or equal to 36, then are determined as uncertain sample, need weight
Rechecked after new extraction nucleic acid；If the sample rechecked still has the amplification of S types, and CT values are less than 40, then judge that sample is the positive, no
It is then feminine gender；If c) sample is without obvious S types amplification curve, but report has CT values, then is determined as non-specific amplification；May be to visit
The discharged non-specific fluorescence signal of pin degraded.Specific decision procedure is with reference to table 1.
The storage life experiment of 3 kit of embodiment
Strong positive 10 is taken respectively5PFU/mL and weakly positive 1036 type of Coxsackie virus A, A10 types, A16 types, the intestines of PFU/mL
The pseudovirus dilution of road 71 types of virus detects sample for assessment, at the 0th day, is distributed into 9 parts and freezes in -70 DEG C of refrigerators
In.The kit that establishment finishes is positioned over -20 DEG C of preservations, takes 0,10,15,30,60,90,120,150,180 and 360 respectively
It kit carries out storage life experiment.Storage life testing result is as shown in table 2：
2 storage life result of the test of table
| Storage life | Detect the validity of four kinds of target viral strong positives and weakly positive sample |
| 0th day | It is effective |
| 10th day | It is effective |
| 15th day | It is effective |
| 30th day | It is effective |
| 60th day | It is effective |
| 90th day | It is effective |
| 120th day | It is effective |
| 150th day | It is effective |
| 180th day | It is effective |
| 360th day | It is effective |
As shown in Table 2, kit is stored in -20 DEG C of refrigerators, and four kinds of target virals and enteron aisle disease are detected in different storage lives
Poison is universal effectively, test result indicates that the storage life of the kit is at least 6 months.
The specific test of 4 kit of embodiment
Select influenza virus, rubella virus, measles virus, Respiratory Syncytial Virus(RSV), mumps virus, parainfluenza virus, B
Race streptococcus, adenovirus, rotavirus, varicellazoster virus (Chinese Center for Disease Control and Prevention virosis prevention and control
There is provided), it is close with detection target viral kind, there are the similar virus of environment as Virus Sample to be checked.
Detect these cause of diseases to be checked using kit of the present invention, in negative control, positive control and the positive Quality Control be into
It is vertical, it was demonstrated that detecting system is set up；Virus to be checked does not occur nonspecific fluorescence signal, shows that kit of the present invention can have
Effect distinguishes non-targeted virus, has preferable specific (result is as shown in figures 13-18).
The minimum detectability experiment of 5 kit of embodiment
Assessment detection sample：It is generation to select 6 type of Coxsackie virus A, A10 types, A16 types, enterovirns type 71, pseudovirus
Table strain, 10 are diluted to by the viral suspension of 4 pseudovirus respectively8PFU/mL.By 4 templates distinguish gradient dilutions into equivalent to
107PFU/mL, 106PFU/mL, 105PFU/mL, 104PFU/mL, 103PFU/mL, 102The detection sample of PFU/mL, 10PFU/mL,
The RNA of these target pseudovirus dilutions is extracted respectively.
6 type of Coxsackie virus A, A10 types, A16 types, enterovirns type 71 pseudovirus are detected respectively using kit of the present invention
The template of different dilution factors.Operation is carried out according to embodiment 2 and result judges, kit detects the minimum inspection of 5 target virals
Rising limit result of the test is as shown in table 3：
3 kit of table detection Coxsackie virus A, 6 type, A10 types, A16 types, enterovirns type 71 and enterovirus universal
Minimum detectability result of the test
As seen from Table 3, the minimum detectability of 5 kinds of target virals of kit detection reaches 102PFU/mL, kit are overall
Minimum detectability be each reaction detection 1 copy target molecule.
Comparative example 1
According to the method for primer sequence same as Example 1, the detection target of kit A, B pipe is adjusted, Coxsack is sick
Malicious A6 types are adjusted to B and managed, and are differed only in, and the primed probe shown in SEQ ID NO.1-3 is adjusted to B and is managed, is obtained to having a competition
Agent box 1.
Wherein, A pipes primed probe：The 10 type sense primer of Coxsackie virus A of nucleotide sequence shown in SEQ ID NO.4,
The 10 type anti-sense primer of Coxsackie virus A of nucleotide sequence shown in SEQ ID NO.5, has nucleotide shown in SEQ ID NO.6
The 10 type probe of Coxsackie virus A of sequence, 5 ' ends of the 10 type probe of Coxsackie virus A are marked with VIC luminophores, 3 ' ends
It is marked with fluorescent quenching group BHQ1；
The coxsackie virus A 16-type sense primer of nucleotide sequence shown in SEQ ID NO.7, core shown in SEQ ID NO.8
The coxsackie virus A 16-type anti-sense primer of nucleotide sequence, has the Coxsackie virus of nucleotide sequence shown in SEQ ID NO.9
A16 type probes, 5 ' ends of the coxsackie virus A 16-type probe are marked with FAM luminophores, and 3 ' ends are marked with fluorescent quenching base
Group BHQ1；
The nucleotide sequence of Quality Control sense primer is as shown in SEQ ID NO.16 in the positive, Quality Control anti-sense primer in the positive
Nucleotide sequence is as shown in SEQ ID NO.17, and the nucleotide sequence of Quality Control probe is as shown in SEQ ID NO.18 in the positive, institute
5 ' the ends for stating Quality Control probe in the positive are marked with ROX luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ2.
B pipe primed probes：The 6 type sense primer of Coxsackie virus A of nucleotide sequence shown in SEQ ID NO.1, SEQ ID
The 6 type anti-sense primer of Coxsackie virus A of nucleotide sequence shown in NO.2, has Ke of nucleotide sequence shown in SEQ ID NO.3
Sa Qi virus A6 type probes, 5 ' ends of the 6 type probe of Coxsackie virus A are marked with CY5 luminophores, and 3 ' ends are marked with fluorescence
Quenching group BHQ3；
The enterovirns type 71 sense primer of nucleotide sequence shown in SEQ ID NO.10, nucleosides shown in SEQ ID NO.11
The enterovirns type 71 anti-sense primer of acid sequence, the enterovirns type 71 with nucleotide sequence shown in SEQ ID NO.12 are visited
Pin, 5 ' ends of the enterovirns type 71 probe are marked with VIC luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ1；
The enterovirus universal sense primer of nucleotide sequence shown in SEQ ID NO.13, core shown in SEQ ID NO.14
The enterovirus universal anti-sense primer of nucleotide sequence, the enterovirus with nucleotide sequence shown in SEQ ID NO.15 are general
Type probe, 5 ' ends of the enterovirus universal probe are marked with FAM luminophores, and 3 ' ends are marked with fluorescent quenching group
BHQ1。
The nucleotide sequence of Quality Control sense primer is as shown in SEQ ID NO.16 in the positive, Quality Control anti-sense primer in the positive
Nucleotide sequence is as shown in SEQ ID NO.17, and the nucleotide sequence of Quality Control probe is as shown in SEQ ID NO.18 in the positive, institute
5 ' the ends for stating Quality Control probe in the positive are marked with ROX luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ2.
Specificity and minimum detectability experiment, the results show contrast agent are carried out according to the method identical with embodiment 4 and 5
For the B pipes of box 1 when detecting enterovirus EV 71 type, minimum detectability is only 103PFU/mL, is worse than the examination set up in embodiment 1
Agent box.
Comparative example 2
Contrast agent box 1-3 is assembled in the same manner as shown in Example 1.The primed probe summary sheet of comparative example is shown in Table 4.
Table 4 contrasts primer sequence table
Differ only in, the primed probe shown in SEQ ID NO.1-6 is replaced with into drawing shown in SEQ ID NO.19-24
Thing and probe obtain contrast agent box 2.
Primer shown in SEQ ID NO.7-12 is replaced with to the primer and probe acquisition pair shown in SEQ ID NO.25-30
Than kit 3.
Primer shown in SEQ ID NO.13-15 is replaced with to the primer and probe shown in SEQ ID NO.31-33 to obtain
Contrast agent box 4.
Specificity and minimum detectability experiment, the results show contrast agent are carried out according to the method identical with embodiment 4 and 5
For box 2,3 and 4 when detecting Respiratory Syncytial Virus(RSV), there is an example non-specific amplification in the A pipe FAM passages of contrast agent box 2, right
The minimum detectability more general than the CA16 and enteron aisle of kit 3 is only 103PFU/mL, there are an example enteron aisle disease for contrast agent box 4
Poison is universal not to be detected.Illustrate that contrast agent box 2,3 and 4 minimum detectabilities, coverage and specificity aspect are worse than implementation
The kit set up in example 1.
It is further to note that each particular technique feature described in above-mentioned embodiment, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should equally be considered as content disclosed in this invention.
The preferred embodiment of the present invention described in detail above, still, during present invention is not limited to the embodiments described above
Detail, in the range of the technology design of the present invention, a variety of simple variants can be carried out to technical scheme, this
A little simple variants belong to protection scope of the present invention.
Sequence table
<110>Beijing Zhuo Cheng Hui Sheng biotech inc
<120>Multiplex PCR detection Coxsackie virus, enterovirns type 71 and enterovirus universal primed probe group
<130> 7078ABT
<160> 33
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 1
gcgaaattga gtgatccacc c 21
<210> 2
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 2
aaattggtag cttgtttgtg ctc 23
<210> 3
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 3
atctgtcccg ttcatgtcgc cag 23
<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 4
ggagtagtta acctcacrga t 21
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 5
ttctgcgttr aacctcatgt at 22
<210> 6
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 6
tgtagcatac ccagtrgtgt ccgtcc 26
<210> 7
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 7
cgggyacaca gaatacagat ggt 23
<210> 8
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 8
aagcgcatgt aggtraataa ct 22
<210> 9
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 9
ccgccgcagc tgagcatatc c 21
<210> 10
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 10
gcytatcaat ggttttatga cgga 24
<210> 11
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 11
gttattaggr catgccccgt att 23
<210> 12
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 12
cccacattcg gagaacacaa acaggag 27
<210> 13
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 13
tgtcgtaacg ggyaactct 19
<210> 14
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 14
ttgtcaccat aagcagccaa tat 23
<210> 15
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 15
acacggacac ccaaagtagt cgg 23
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 16
tgtgagggta aacaactggc 20
<210> 17
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 17
tgcgacctga gcaacaac 18
<210> 18
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 18
tgctgctggc taccctgtgg aacacc 26
<210> 19
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 19
agaaycttat tgagactcgc tgt 23
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 20
gctagtgccc gagtccttca 20
<210> 21
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 21
cctaccagcc ctgcacgaga g 21
<210> 22
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 22
tacaaatgtt gaatccgcag ccaa 24
<210> 23
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 23
aaccacacaa cgggtytc 18
<210> 24
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 24
accactccca gctcacaccg at 22
<210> 25
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 25
ayttcacatt tgtcgtagcc aa 22
<210> 26
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 26
tctgccaagc aaacgaatct c 21
<210> 27
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 27
ccaggggctc cgaaacctac ttcc 24
<210> 28
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 28
ttaactcrca cagtacagct ga 22
<210> 29
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 29
gcgcgtaacc tgttatatct atg 23
<210> 30
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 30
accatttggg ttagttgtgc cctc 24
<210> 31
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 31
tgcggctaat cccaactg 18
<210> 32
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 32
ttgtcaccat aagcagcyaa tata 24
<210> 33
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 33
actctgcagc ggaaccgact ac 22
Claims (9)
1. 6 types of multiplex PCR detection Coxsackie virus A/A10 types/A16 types/enterovirns type 71/enterovirus universal primer
Probe groups, it is characterised in that including component A and component B,
Wherein, the component A includes：
The 6 type sense primer of Coxsackie virus A of nucleotide sequence shown in SEQ ID NO.1,
The 6 type anti-sense primer of Coxsackie virus A of nucleotide sequence shown in SEQ ID NO.2,
6 type probe of Coxsackie virus A with nucleotide sequence shown in SEQ ID NO.3, the 6 type probe of Coxsackie virus A
5 ' ends be marked with CY5 luminophores, 3 ' ends are marked with fluorescent quenching group BHQ3；
The 10 type sense primer of Coxsackie virus A of nucleotide sequence shown in SEQ ID NO.4,
The 10 type anti-sense primer of Coxsackie virus A of nucleotide sequence shown in SEQ ID NO.5,
10 type probe of Coxsackie virus A with nucleotide sequence shown in SEQ ID NO.6,10 type of Coxsackie virus A are visited
5 ' ends of pin are marked with VIC luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ1；
The coxsackie virus A 16-type sense primer of nucleotide sequence shown in SEQ ID NO.7,
The coxsackie virus A 16-type anti-sense primer of nucleotide sequence shown in SEQ ID NO.8,
Coxsackie virus A 16-type probe with nucleotide sequence shown in SEQ ID NO.9, the coxsackie virus A 16-type are visited
5 ' ends of pin are marked with FAM luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ1；
The component B includes：
The enterovirns type 71 sense primer of nucleotide sequence shown in SEQ ID NO.10,
The enterovirns type 71 anti-sense primer of nucleotide sequence shown in SEQ ID NO.11,
Enterovirns type 71 probe with nucleotide sequence shown in SEQ ID NO.12, the 5 ' of the enterovirns type 71 probe
End is marked with VIC luminophores, and 3 ' ends are marked with fluorescent quenching group BHQ1；
The enterovirus universal sense primer of nucleotide sequence shown in SEQ ID NO.13,
The enterovirus universal anti-sense primer of nucleotide sequence shown in SEQ ID NO.14,
Enterovirus universal probe with nucleotide sequence shown in SEQ ID NO.15, the enterovirus universal probe
5 ' ends be marked with FAM luminophores, 3 ' ends are marked with fluorescent quenching group BHQ1.
2. primed probe group according to claim 1, wherein, also respectively containing Quality Control in the positive in the component A and component B
Primed probe,
The nucleotide sequence of Quality Control sense primer is as shown in SEQ ID NO.16 in the positive,
The nucleotide sequence of Quality Control anti-sense primer is as shown in SEQ ID NO.17 in the positive,
The nucleotide sequence of Quality Control probe is as shown in SEQ ID NO.18 in the positive, 5 ' end marks of Quality Control probe in the positive
There are ROX luminophores, 3 ' ends are marked with fluorescent quenching group BHQ2.
A kind of 3. 6 types of multiple fluorescence quantitative PCR detection Coxsackie virus A/A10 types/A16 types/enterovirns type 71/enterovirus
Universal detection method, it is characterised in that the detection method includes the following steps：
(1) total nucleic acid of sample to be tested is extracted；
(2) using the total nucleic acid as pcr template, using the primed probe group described in claim 1 or 2 to the pcr template into
Row multiple fluorescence quantitative PCR expands；
Wherein, the multiple fluorescence quantitative PCR carries out in two systems at the same time,
In first system, the first amplification is carried out to the pcr template using the component A,
In second system, the second amplification is carried out to the pcr template using the component B；
(3) respectively collect first system in FAM, VIC, CY5 and ROX fluorescence channel signal, collect second system in VIC,
FAM and ROX fluorescence channel signals.
4. detection method according to claim 3, wherein, the detection method further includes adds in two systems respectively
Quality Control pET28a plasmid templates in the positive.
5. the detection method according to claim 3 or 4, wherein, in the primed probe group, the final use of every primer
Concentration is respectively 0.3-0.6 μM, and the final concentration of every probe is respectively 0.1-0.3 μM.
6. detection method according to claim 5, wherein, the reaction system of two multiple fluorescence quantitative PCRs is including following
The each component of content：
Hotstar archaeal dna polymerases containing 1-3U, the RNase inhibitor of 20-60U, the MMLV of 100-300U in 20 μ L reaction solutions
Reverse transcriptase, the Tris-HCl buffer solutions that concentration is 20mM and pH is 8.3, the pET28a, 0.2- of 0.00003-0.0003ng/ μ L
0.4mM dNTP, 2-6mM MgCl2。
7. detection method according to claim 6, wherein, the reaction condition of multiple fluorescence quantitative PCR includes the following steps：
a：45℃18-22min；
b：95℃4-6min；
c：95℃15-60s；
d：50-60 DEG C of 15-60s, c-d circulate 45 reactions.
8. 6 types of multiple fluorescence quantitative PCR detection Coxsackie virus A/A10 types/A16 types/enterovirns type 71/enterovirus is general
The kit of type, it is characterised in that the kit includes the primed probe group described in claim 1 or 2.
9. kit according to claim 8, wherein, the final concentration of every primer is respectively in the kit
0.3-0.6 μM, the final concentration of every probe is respectively 0.1-0.3 μM.
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| CN111534637A (en) * | 2020-04-27 | 2020-08-14 | 江苏派森杰生物科技有限公司 | Universal primer, probe and kit for enterovirus nucleic acid detection |
| CN112593011A (en) * | 2020-12-25 | 2021-04-02 | 中山大学 | Primer and probe for detecting coxsackie virus B group |
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| CN116286803A (en) * | 2023-02-07 | 2023-06-23 | 广州凯普医药科技有限公司 | Detection primer probe combination and gene chip for synchronously detecting 22 hand-foot-mouth viruses |
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Application publication date: 20180515 |