CN106191316B - Multiplex PCR detects seven kinds of diarrhea virus primer sets and kit and its detection method - Google Patents
Multiplex PCR detects seven kinds of diarrhea virus primer sets and kit and its detection method Download PDFInfo
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Abstract
The present invention provides multiplex PCRs to detect seven kinds of diarrhea virus primer sets, wherein the primer sets include primer shown in SEQ ID NO.1 and 3-16.The present invention also provides the kits that a kind of multiplex PCR detects 7 kinds of diarrhea virus, and the kit includes multiple PCR primer group, reverse transcriptase and archaeal dna polymerase of the present invention.It is of the invention through the above technical solution to be improved significantly to seven kinds of diarrhea virus while the sensibility, specificity and the simplicity that are detected.
Description
Technical field
The present invention relates to field of biotechnology, and in particular, to multiplex PCR detects seven kinds of diarrhea virus primer sets and examination
Agent box and its detection method.
Background technique
Diarrhea virus infection is the common disease for endangering human health.In developing country, it has also become lead to Infant and child deaths
Etiological.Wherein, rotavirus A group, norovirus GI type, norovirus GII type, letter such as virus, astrovirus, enteron aisle
Adenovirus, bocavirus etc. are the most common reasons for causing diarrhea.And it is different from diarrhea caused by bacterium, helminth, for disease
Virus diarrhea there is no effective drug therapy at present.But it since clinically the habitual of antibiotic is applied, does not simply fail to kill abdomen
Diarrhea virus can also kill the normal flora in enteron aisle, cause Flora Disturbance and then extend the diarrhea time.To virus diarrhea
Prevention and control in, vaccine inoculation is most potential method, it is therefore necessary to be had adequately to the epidemiologic feature of virus diarrhea
Understanding.So there is an urgent need to easy, quick, automation diarrhea virus multiple detection methods.
Because most of diarrhea virus cannot be separately cultured, so mainly using morphology, immunology and molecular biology
Method is detected.Initially, the identification of diarrhea virus relies primarily on morphologic method, such as Electronic Speculum.But there are instruments for electron microscopy
Equipment valuableness, the problem of sensibility difference.With the development of immunology and molecular biology method, occurs a series of enzymes successively and exempt from
Epidemic disease and PCR method are used for the identification of diarrhea virus.Wherein, for the detection of rotavirus A group, colloidal gold is still mainly used at present
Technology is detected, but colloidal gold detection has that sensitivity is low, false positive is high.For other diarrhea virus, more adopt
It is detected with nest-type PRC and substance real time fluorescent PCR method.Though nest-type PRC has compared with hypersensitivity, because using two-wheeled PCR
Target to be checked is expanded, there are higher laboratory pollution risks.Substance real time fluorescent PCR method is needed to a series of
Diarrhea virus is detected one by one, time-consuming and laborious.
There are also the reports using multiple PCR technique screening diarrhea correlated virus at present, as Chinese Patent Application No. is
The patent of invention of CN201310033219.7 discloses kit and its detection side of a kind of 13 kinds of diarrhea virus of synchronous detection
Method.Its main feature is that reverse transcription primer includes a kind of ten RT amplimers of diarrhea RNA virus and people RNA internal reference, and discloses and draw
Object sequence.But detection needs realize result judgement on GeXP genetic analyzer, and the requirement to instrument platform is high, and easily occur
Non-specific band disturbs the judgement of result.And the patent separately carries out reverse transcription and PCR reaction, increases operation step
The rapid and reaction time.It is unfavorable for the accurate result judgement of laboratory quick obtaining.
Chinese Patent Application No. is that the patent of invention of CN20131012328.9 discloses a kind of diarrhea virus detection kit
And its method, it further comprises for such as viral 1 type of letter and 2,4 types, 1 type of norovirus, 2 type of norovirus, rotavirus, adenopathy
Poison, the primer pair of astrovirus and probe sequence, can disposably detect wherein virus relevant to diarrhea.But this method is using spy
The method of needle composite coding cannot effectively be distinguished the mixed infection of different diarrhea virus and substance infection.Such as promise such as disease
Malicious 2 types infection has two kinds of signals of FAM and HEX while discharging, but if sample is the dual sense of letter such as virus and rotavirus
Dye, then can equally release two kinds of signals of FAM and HEX, this carries out the real-time fluorescence skill of result judgement for relying on fluorescence signal
Art platform, it may appear that the problem that result can not accurately determine.
Some other report about diarrhea virus detection, it is generally existing to fail to fully consider the main popular type in China
The problem of, the problem of design of primer and (or probe) can not cover object to be measured comprehensively, false negative result causes
The problem of recall rate significantly reduces.
Therefore the defects of detection mode of existing diarrhea virus is lower there are complicated for operation and specific and sensibility.
Summary of the invention
It is an object of the invention to solve the costly, time-consuming length of instrument platform present in existing diarrhea virus detection technique,
The problem that sensibility and specificity is poor, recall rate is low and coverage is poor provides a kind of new diarrhea virus detection primer and method.
To achieve the above objectives, the present invention provides a kind of multiplex PCRs to detect seven kinds of diarrhea virus primer sets, wherein
The primer sets include primer shown in SEQ ID NO.1 and 3-16；Seven kinds of diarrhea virus include rotavirus A group, promise such as
Viral GI type, norovirus GII type, letter such as virus, astrovirus, intestinal adenovirus and bocavirus.
The present invention also provides the detection method that a kind of multiplex PCR detects seven kinds of diarrhea virus, this method includes following step
It is rapid:
(1) total nucleic acid of sample to be tested is extracted；
(2) reverse transcription is carried out to the total nucleic acid and obtains pcr template；With primer sets of the present invention to the PCR mould
Plate carries out multiplexed PCR amplification, the material after obtaining multiplexed PCR amplification；
(3) nucleic acid electrophoresis detection is carried out to the material after the multiplexed PCR amplification, obtains the result of nucleic acid electrophoresis detection；
If the amplification containing 139bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain rotavirus A group in the sample to be tested；
If the amplification containing 364bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain norovirus GI type in the sample to be tested；
If the amplification containing 487bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain norovirus GII type in the sample to be tested；
If the amplification containing 415bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates in the sample to be tested containing letter such as virus；
If the amplification containing 282bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain astrovirus in the sample to be tested；
If the amplification containing 200bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain intestinal adenovirus in the sample to be tested；
If the amplification containing 166bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain bocavirus in the sample to be tested.
The present invention also provides the kits of 7 kinds of diarrhea virus of detection, and the kit includes of the present invention multiple
PCR primer group, reverse transcriptase and archaeal dna polymerase；Seven kinds of diarrhea virus include rotavirus A group, norovirus GI type, promise
Such as viral GII type, letter such as virus, astrovirus, intestinal adenovirus and bocavirus.
On the other hand, the present invention also provides primer sets as described above in the kit for preparing seven kinds of diarrhea virus of detection
In purposes；Wherein, seven kinds of diarrhea virus include rotavirus A group, norovirus GI type, norovirus GII type, letter such as
Virus, astrovirus, intestinal adenovirus and bocavirus.
The present invention establishes common seven kinds of diarrhea virus multiple PCR detection primer groups and detection side through the above technical solution
Method, can be realized morphology, immunology and substance real-time fluorescence detection institute it is impossible quickly, comprehensively, it is sensitive, special,
Automatic result judgement is improved significantly to seven kinds of diarrhea virus while the sensibility, specificity and the simplicity that are detected.
Other features and advantages of the present invention will the following detailed description will be given in the detailed implementation section.
Detailed description of the invention
The drawings are intended to provide a further understanding of the invention, and constitutes part of specification, with following tool
Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 be after the total nucleic acid of 16 kinds of unrelated pathogen samples is mixed with the positive internal reference of equivalent in embodiment 1 according to
Method provided by the present invention is detected, and all the positive internal reference band of 532bp has been arrived in amplification to multiplex PCR, but removes 532bp
Positive internal reference band outside, do not amplify the Capillary Electrophoresis map of other bands；
Wherein, M is Size Marker, and 1 is that the total nucleic acid of 16 kinds of unrelated pathogen samples and the positive internal reference of equivalent mix
Close sample.
Fig. 2 be 37 hypotypes in embodiment 1 standard items plasmid under 20 copies/μ L concentration (with the concentration of pcr template
Meter) it can amplify the Capillary Electrophoresis map of corresponding purpose band and positive internal reference band；
Wherein, M is Size Marker, and swimming lane 1 is rotavirus A group standard items plasmid, and swimming lane 2 is norovirus GI type
Standard items plasmid, swimming lane 3 are norovirus GII type standard items plasmid, and swimming lane 4 is letter such as Virus Standard quality grain, and swimming lane 5 is star
Shape Virus Standard quality grain, swimming lane 6 are intestinal adenovirus standard items plasmid, and swimming lane 7 is bocavirus standard items plasmid, swimming lane 8
Amount to the standard items plasmid of 37 hypotypes and the mixture of positive internal reference for 7 kinds of diarrhea virus.
Fig. 3 is that specificity, sensibility and the coverage rate of the comparison primer sets 1-7 of comparative example 1 are worse than the primer of embodiment 1
The Capillary Electrophoresis map of group；
Wherein, M is Size Marker, and swimming lane 1 is rotavirus A group standard items plasmid, and swimming lane 2 is norovirus GI type
Standard items plasmid, swimming lane 3 are norovirus GII type standard items plasmid, and swimming lane 4 is letter such as Virus Standard quality grain, and swimming lane 5 is star
Shape Virus Standard quality grain, swimming lane 6 are intestinal adenovirus standard items plasmid, and swimming lane 7 is bocavirus standard items plasmid, swimming lane 8
Amount to the standard items plasmid of 37 hypotypes and the mixture of positive internal reference for 7 kinds of diarrhea virus, swimming lane 9 is 16 kinds of unrelated cause of diseases
The mixing sample of the positive internal reference of the total nucleic acid and equivalent of body sample.
Specific embodiment
Below in conjunction with attached drawing, detailed description of the preferred embodiments.It should be understood that this place is retouched
The specific embodiment stated is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
The present invention provides a kind of multiplex PCRs to detect seven kinds of diarrhea virus primer sets, wherein the primer sets include SEQ
Primer shown in ID NO.1 and 3-16；Seven kinds of diarrhea virus include rotavirus A group (Rotavirus, group A), promise
Such as viral GI type (Norovirus-GI), norovirus GII type (Norovirus-GII), letter such as virus (Sapovirus), star
Shape virus (Astrovirus), intestinal adenovirus (Adenovirus) and bocavirus (Bocavirus).
Wherein it is preferred to which the primer sets further include primer shown in SEQ ID NO.17-18.SEQ ID NO.17-18
Shown in primer be using pET28a plasmid as the pair of primers of stencil design, as positive internal reference.
Multiple PCR primer group detection target gene of the invention includes the VP6 gene of rotavirus A group, norovirus GI
The orf1-orf2 gene of type, the orf1-orf2 gene of type G 2 norovirus, letter such as the orf1-orf2 gene of virus, starlike disease
The orf1a gene of poison, the Hexon gene of intestinal adenovirus, bocavirus Ns1 gene.Specifically, diarrhea virus multiplex PCR
Type/the hypotype for detecting target gene and Major Epidemic strain is as shown in table 1.
Type/hypotype of 1 diarrhea virus multiplex PCR of table detection target gene and Major Epidemic strain
It is (homologous general based on catenation sequence containing LHP sequence in the sequence of primer shown in SEQ ID NO.3-18
Primer (based on ligation-sequence homogenous Primer, LHP).LHP sequence such as SEQ ID NO.2 institute
Show.Specifically, the information of SEQ ID NO.1-18 is as shown in table 2.
2 primer information table of table
Wherein, SEQ ID NO.1 is homologous universal primer, which is connected with catenation sequence CATTAA, constitutes LHP sequence
(based on catenation sequence homologous universal primer (based on ligation-sequence homogenous Primer,
LHP))。
The present invention also provides the detection methods of seven kinds of diarrhea virus, and this method comprises the following steps:
(1) total nucleic acid of sample to be tested is extracted；
(2) reverse transcription is carried out to the total nucleic acid and obtains pcr template；With primer sets of the present invention to the PCR mould
Plate carries out multiplexed PCR amplification, the material after obtaining multiplexed PCR amplification；
(3) nucleic acid electrophoresis detection is carried out to the material after the multiplexed PCR amplification, obtains the result of nucleic acid electrophoresis detection；
If the amplification containing 139bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain rotavirus A group in the sample to be tested；
If the amplification containing 364bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain norovirus GI type in the sample to be tested；
If the amplification containing 487bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain norovirus GII type in the sample to be tested；
If the amplification containing 415bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates in the sample to be tested containing letter such as virus；
If the amplification containing 282bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain astrovirus in the sample to be tested；
If the amplification containing 200bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain intestinal adenovirus in the sample to be tested；
If the amplification containing 166bp produces in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection
Object then indicates to contain bocavirus in the sample to be tested.
Wherein it is preferred to the final of primer shown in SEQ ID NO.3-16 is respectively 0.1-0.8 μM using concentration, SEQ
The final use concentration of primer shown in ID NO.1 is 0.4-0.8 μM.
Wherein it is preferred to which the reaction condition of step (2) includes the program of following a-i:
A:49-51 DEG C, 28-32min；
B:94-96 DEG C, 13-18min；
C:94-96 DEG C, 28-32s,
D:54-56 DEG C, 28-32s,
E:71-73 DEG C, 58-62s, carry out the circulation of 10 c-e；
F:94-96 DEG C, 28-32s,
G:49-51 DEG C, 28-32s,
H:71-73 DEG C, 59-61s, carry out the circulation of 30 f-h；
I:71-73 DEG C, 5-10min.
Wherein, in above procedure, program a is to carry out the process that reverse transcription obtains pcr template to total nucleic acid, reverse transcription
It is cDNA that purpose, which is by the RNA reverse transcription in total nucleic acid, and the DNA in process of reverse-transcription in total nucleic acid is unaffected,
After process of reverse-transcription, the DNA in cDNA and total nucleic acid that reverse transcription obtains carries out the more of next step collectively as pcr template
Weight PCR amplification.
In the method provided by the disclosure, the constituent of reaction system includes the total nucleic acid of sample to be tested, multiplex PCR
Primer sets, reverse transcriptase, archaeal dna polymerase, PCR reaction buffer and dNTP carry out reverse transcription mistake in the same reaction system
Journey and multiplexed PCR amplification reaction.
Wherein, the sample to be tested can include but is not limited in food, drug, excreta, vomitus and body fluid extremely
Few one kind.Preferably, the detection method that multiplex PCR of the invention detects seven kinds of diarrhea virus is not used in diagnosis.In other words, seven kinds
Whether the qualitative and quantitative result of diarrhea virus occurs without one-to-one correlation, that is, seven kinds of diarrhea virus with diarrhea
Qualitative and quantitative result be not belonging to diagnostic result, but the testing result of the qualitative and quantitative of seven kinds of diarrhea virus being capable of conduct
Average information, for reference for clinicians.
Wherein it is preferred to which the nucleic acid electrophoresis detection includes nucleic acid gel electrophoresis and/or nucleic acid Capillary Electrophoresis.
In one embodiment of the invention, divided as a result using the full-automatic capillary electrophoresis analysis instrument of QIaxcel
The platform of analysis.The intelligent interpretation of result program of full automatic working and setting based on QIaxcel, to realize the automatic of result
Decision analysis.
The present invention also provides the kit that a kind of multiplex PCR detects 7 kinds of diarrhea virus, the kit includes the present invention
Multiple PCR primer group, reverse transcriptase and the archaeal dna polymerase；Seven kinds of diarrhea virus include rotavirus A group, promise such as
Viral GI type, norovirus GII type, letter such as virus, astrovirus, intestinal adenovirus and bocavirus.
Wherein it is preferred to which the kit further includes PCR reaction buffer and dNTP.
On the other hand, the present invention also provides primer sets as described above in the kit for preparing seven kinds of diarrhea virus of detection
In purposes；Wherein, seven kinds of diarrhea virus include rotavirus A group, norovirus GI type, norovirus GII type, letter such as
Virus, astrovirus, intestinal adenovirus and bocavirus.
Particularly, technical solution of the present invention can achieve following detection effect:
(1) Multiple detection
Detection method established by the present invention can identify the relevant seven kinds of virus of diarrhea in a PCR reaction, quickly
Testing result is obtained, time, cost of human and material resources are saved.
(2) specificity is high
The specificity of detection method established by the present invention is mainly reflected in the specificity of a whole set of specific primer, and
The validity of full-automatic result judgement.All primers all pass through blast and compare analysis, conservative and specificity with height；
It can be good at distinguishing, living environment identical bacterium close with detection target species symbolic animal of the birth year and disease by specificity experiments verifying simultaneously
Poison, including artificial tuberculosis yersinia genus, escherichia coli, Enterobacter sakazakii, Shigella, staphylococcus aureus, sramana
Salmonella, vibrio parahemolyticus, Listeria monocytogenes, comma bacillus, bacillus cereus, campylobacter jejuni,
Campylobacter Coli, people's enteric coronavirus virus, human parechovirus, bed ripples virus, Coxsackie virus etc., it was demonstrated that detection method has
Non-detection target can be accurately distinguished and be come by the specificity of height.
(3) high sensitivity
Detection while detection method established by the present invention can be realized 7 targets, it is every in each reaction system
The detection sensitivity of a detection target can reach 20 copies/μ L.
(4) cost is relatively low
Multi-PCR detection method established by the present invention reduces human cost and time cost in operability, originally
Substance detection needs 7 artificial and 7 times of times, only needs 1 artificial and 1 reaction time using the method now；This is more
Re-detection method saves the reagent consumption that repetition detects the same sample simultaneously, and maximum can save 50% reagent cost.
(5) time is saved
The reaction of traditional two-step method, takes a long time.The present invention uses the RT-PCR system of one-step method, including extracting
Whole experiment process only need 3 hours, significantly shorten detection needed for time.
(6) covering is comprehensive
Method established by the present invention can cover the main diarrhea virus epidemic strain in China comprehensively, can be to rotavirus A group
G1P [8], G2P [4], G3P [8], G4, G9P [8], G10, G12 type, norovirus G1.4, G1.5, G1.2, G1.3 type, promise is such as
Viral GII.4, GII.3, GII.6, GII.12, GII.14, GII.2, GII.16, GII.d, GII.b type, letter such as virus GI.1,
GI.2, GI.3, GI.5, GI.8, GII.1, GIV type, astrovirus HAstV-1a, HAstV-1b, HAstV-1d, HAstV-2,
HAstV-4 type, intestinal adenovirus Adv40, Adv41 type, bocavirus HBoV-1, HBoV-2, HBoV-4 type etc. are detected.
(7) prevent false negative result
The positive internal reference added in reaction system of the present invention can be prompted effectively because of operation error, PCR mortifier
Etc. false negative testing result caused by reasons.
The present invention provides whole solution for diarrhea virus rapid screening, is able to achieve the fast of common diarrhoea virus
Speed, automation decision analysis.
Hereinafter, present invention will be further described in detail through examples.
1, primer synthesizes:
According to sequence shown in table 3, the synthesis of the primer of SEQ ID NO.1 and 3-18 is carried out.
3 primer sequence table of table
As unit of the amount of substance, the primer of SEQ ID NO.3-18 is respectively taken into 1 parts by weight, the SEQ ID with 4 parts by weight
The primer of NO.1 mixes, and constitutes the primer sets of the present embodiment.Then specificity verification is carried out to above-mentioned primer sets and sensibility is tested
Card.
2, specificity verification:
Select following 16 kinds of unrelated pathogen to interfere sample as simulation: artificial tuberculosis yersinia genus is (purchased from Chinese medicine
Culture Collection Center, number 53501), escherichia coli (be purchased from Chinese medicine Culture Collection Center, number 44101),
Enterobacter sakazakii (being purchased from Chinese medicine Culture Collection Center, number 21665), Shigella (protect purchased from Chinese medicine strain
Hiding center, number 51054), staphylococcus aureus (be purchased from Chinese medicine Culture Collection Center, number 26003), sramana
Salmonella (being purchased from Chinese medicine Culture Collection Center, number 50001), vibrio parahemolyticus (are purchased from Chinese medicine culture presevation
Center, number 21617), the Hypertrophic listeria spp of monokaryon (be purchased from Chinese medicine Culture Collection Center, number 54002),
Comma bacillus (being purchased from Chinese medicine Culture Collection Center, number 1109), bacillus cereus (protect purchased from Chinese medicine strain
Hiding center, number 63303), campylobacter jejuni (be purchased from Chinese medicine Culture Collection Center, number 33291), colon bending
The diarrhea Related Bacterias such as bacterium (being purchased from Chinese medicine Culture Collection Center, number 52204) and the viral (source of people's enteric coronavirus
From national CDC, number is IVDC 2.2334), human parechovirus's (be originated from country CDC, number is IVDC 2.2335), bed ripples
Virus (being originated from country CDC, number is IVDC 2.2336), Coxsackie virus (are originated from Chinese Academy of Sciences Wuhan virus institute, number
For IVCAS 6.0190).
Above-mentioned simulation interference sample carries out the extraction of total nucleic acid, every kind of sample extraction obtains for specificity assessment respectively
Total nucleic acid be dissolved in TE buffer, concentration be 1ng/ μ L.
It is mixed using the total nucleic acid of every kind of sample obtained above with the positive internal reference (pET28a plasmid) of equivalent, as
Template carries out reverse transcription and multiplexed PCR amplification with primer sets obtained above.
The preparation program of multi-PRC reaction system includes: the 2 μ L of template by taking 25 μ L systems as an example, SEQ ID in primer sets
Final concentration of 0.2 μM of the primer of NO.3-18, final concentration of 0.8 μM of the primer of SEQ ID NO.1, reverse transcriptase, archaeal dna polymerase,
PCR buffer and dNTP and MgCl2Reference enzyme specification uses.
The condition of reaction includes the steps that following a-i: a:50 DEG C of 30min；B:95 DEG C of 15min；C:95 DEG C of 15-60s, d:
50-65 DEG C of 15-60s, e:72 DEG C of 30-120s, c-e recycle 10 reactions；F:95 DEG C of 15-60s, g:45-55 DEG C of 15-60s, h:72
DEG C 30-120s, f-h recycle 30 reactions；I:72 DEG C of 5-10min.
Multiple PCR products are placed in the full-automatic capillary electrophoresis analysis instrument of QIAxcel and are analyzed, the results show that 16 kinds
The total nucleic acid of unrelated pathogen sample mixes the multiplex PCR whole amplification carried out as template with the positive internal reference of equivalent and arrives
The positive internal reference band of 532bp, but in addition to the positive internal reference band of 532bp, other bands are not amplified, specifically
As a result as shown in Figure 1.Thus it proves, the primer sets of the present embodiment are difficult to be interfered by unrelated pathogen, with higher special
Property.
3, sensibility is verified:
The standard items plasmid that 7 kinds of diarrhea virus amount to 37 hypotypes is obtained from Chinese Center for Disease Control and Prevention according to table 4.
The standard items plasmid information table of 4 diarrhea virus of table
Above-mentioned standard quality grain is diluted to 106Copy/μ L concentration, then carry out gradient dilution and respectively with equivalent
Positive internal reference mixing, the template as sensitivity assessment；Wherein, gradient includes 106Copy/μ L, 105Copy/μ L, 104It copies
Shellfish/μ L, 103Copy/μ L, 102Copy/μ L, 50 copies/μ L, 20 copies/μ L and 10 copies/μ L.
Multiplexed PCR amplification is carried out with primer sets obtained above.The preparation program of multi-PRC reaction system includes: with 25 μ
For L system, 2 μ L of template, the primer final concentration of SEQ ID NO.3-18 is respectively 0.2 μM in primer sets, SEQ ID NO.1's
Final concentration of 0.8 μM of primer, reverse transcriptase, archaeal dna polymerase, PCR buffer and dNTP and MgCl2Reference enzyme specification makes
With.
The condition of reaction includes the steps that following a-i: a:50 DEG C of 30min；B:95 DEG C of 15min；C:95 DEG C of 30s, d:55 DEG C
30s, e:72 DEG C of 60s, c-e recycle 10 reactions；F:95 DEG C of 30s, g:50 DEG C of 30s, h:72 DEG C of 60s, f-h recycle 30 reactions；
I:72 DEG C of 8min.
Multiple PCR products are placed in the full-automatic capillary electrophoresis analysis instrument of QIAxcel and are analyzed, the results show that above-mentioned
The standard items of 37 hypotypes can amplify corresponding purpose band and positive internal reference item under 20 copies/μ L concentration
Band, specific typical consequence are as shown in Figure 2；Thus prove that the detection sensitivity of the primer sets of the present embodiment can reach 20 copies/μ
The level of L, and the primer sets of the present embodiment can cover 7 kinds of diarrhea virus and amount to 37 hypotypes.In Fig. 2, specifically, the 1st
Also there is the pcr amplification product of 1 139bp in swimming lane, is the amplified band of rotavirus A group；Also there is 1 article of 364bp in 2nd swimming lane
Pcr amplification product, be the amplified band of norovirus GI type；Also there is the pcr amplification product of 1 article of 487bp in 3rd swimming lane, is
The amplified band of norovirus GII type；Also there is the pcr amplification product of 1 article of 415bp in 4th swimming lane, is the amplification item of letter such as virus
Band；Also there is the pcr amplification product of 1 article of 282bp in 5th swimming lane, is the amplified band of astrovirus；6th swimming lane also occurs 1 article
The pcr amplification product of 200bp is the amplified band of intestinal adenovirus；Also there is the pcr amplification product of 1 article of 166bp in 7th swimming lane,
It is the amplified band of bocavirus；8th swimming lane is the standard items plasmid and positive internal reference that 7 kinds of diarrhea virus amount to 37 hypotypes
The amplified band of mixture.
Comparative example 1
Comparison primer sets 1-7 is prepared according to the method for embodiment 1, is shown in Table 5.
Table 5 compares primer sequence table
| SEQ ID NO | Primer pair sequence (5 ' -3 ') |
| 19 | CCC GTC GAT AAA TAT CT GCT CTA TAT TTA ATN CTT YTT TTA GRG |
| 20 | CCC GTC GAT AAA TAT CT GCT CTA TAT TAT TAA CAC TTC TTT TGA A |
| 21 | CCC GTC GAT AAA TAT CT GCT CTA TAT AAA GGT CGC CGC GAA CAG |
| 22 | CCC GTC GAT AAA TAT CT GCT CTA TAT ACC TCC GAG TCC TGC GGC |
| 23 | CCC GTC GAT AAA TAT CT GCT CTA TAT CTA CCA TCT GGC GTG CCT TGC ACC |
| 24 | CCC GTC GAT AAA TAT CT GCT CTA TAT TCC AGC AGG TTG GTT TGG AAA |
| 25 | CCC GTC GAT AAA TAT CT GCT CTA TAT AAC TTA TGC TAA ACT TGC |
| 26 | CCC GTC GAT AAA TAT CT GCT CTA TAT AGA TCC GTG ATG CTA ATG |
| 27 | CCC GTC GAT AAA TAT CT GCT CTA TAT GGT CAA TGA GTT CGT AGT TAT |
| 28 | CCC GTC GAT AAA TAT CT GCT CTA TAT GGC AGG ACG CCT CGG AGT ACC |
| 29 | CCC GTC GAT AAA TAT CT GCT CTA TAT AAC AAG TTC AGA AAC CCC |
| 30 | CCC GTC GAT AAA TAT CT GCT CTA TAT ACA ACC GGG TTT TGG |
| 31 | CCC GTC GAT AAA TAT CT GCT CTA TAT AAA TCT ATT AGT TTA TGA |
| 32 | CCC GTC GAT AAA TAT CT GCT CTA TAT CCT GTA ATT ATA TCC ACT |
Difference is only that, primer shown in SEQ ID NO.3-4 in the primer sets of embodiment 1 is replaced with SEQ ID
Primer shown in NO.19-20 obtains comparison primer sets 1.
Primer shown in SEQ ID NO.5-6 in the primer sets of embodiment 1 is replaced with shown in SEQ ID NO.21-22
Primer obtains comparison primer sets 2.
Primer shown in SEQ ID NO.7-8 in the primer sets of embodiment 1 is replaced with shown in SEQ ID NO.23-24
Primer obtains comparison primer sets 3.
Primer shown in SEQ ID NO.9-10 in the primer sets of embodiment 1 is replaced with shown in SEQ ID NO.25-26
Primer obtain comparison primer sets 4.
Primer shown in SEQ ID NO.11-12 in the primer sets of embodiment 1 is replaced with shown in SEQ ID NO.27-28
Primer obtain comparison primer sets 5.
Primer shown in SEQ ID NO.13-14 in the primer sets of embodiment 1 is replaced with shown in SEQ ID NO.29-30
Primer obtain comparison primer sets 6.
Primer shown in SEQ ID NO.15-16 in the primer sets of embodiment 1 is replaced with shown in SEQ ID NO.31-32
Primer obtain comparison primer sets 7.
Primer shown in SEQ ID NO.3-16 in the primer sets of embodiment 1 is replaced with shown in SEQ ID NO.19-32
Primer obtain comparison primer sets 8.
Wherein, the 1st Lane Sample is rotavirus A group standard items plasmid, and primer is comparison primer sets 1；2nd Lane Sample
For norovirus GI type standard items plasmid, primer is comparison primer sets 2；3rd Lane Sample is norovirus GII type standard quality
Grain, primer are comparison primer sets 3；4th Lane Sample is letter such as Virus Standard quality grain, and primer is comparison primer sets 4；5th swimming
Road sample is astrovirus standard items plasmid, and primer is comparison primer sets 5；6th Lane Sample is intestinal adenovirus standard quality
Grain, primer are comparison primer sets 6；7th Lane Sample is bocavirus standard items plasmid, and primer is comparison primer sets 7；8th swimming
Road sample is the standard items plasmid and positive internal reference mixture that 7 kinds of diarrhea virus amount to 37 hypotypes, and primer is comparison primer
Group 8.Swimming lane 9 is the mixing sample of the total nucleic acid of 16 kinds of unrelated pathogen samples and the positive internal reference of equivalent, and primer is comparison
Primer sets 8.
Specificity and sensitivity tests are carried out in the same manner as shown in Example 1, and typical result is as shown in figure 3, knot
Fruit shows that specificity, sensibility and the coverage rate of the primer 19-32 of this comparative example are worse than the primer of embodiment 1.
It is as shown in Figure 3: in 1-7 swimming lane, the 1st, 2, the corresponding detection target of 6 swimming lanes do not detect；9th swimming lane it is special
Property show in, in addition to positive internal reference other than also remaining miscellaneous band, illustrate that its specificity is poor.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can
No further explanation will be given for the combination of energy.
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 also be regarded as the disclosure of the present invention.
Claims (9)
1. a kind of multiplex PCR detects seven kinds of diarrhea virus primer sets, wherein the primer sets include SEQ ID NO.1 and 3-16
Shown in primer；Seven kinds of diarrhea virus include rotavirus A group, norovirus GI type, norovirus GII type, letter such as disease
Poison, astrovirus, intestinal adenovirus and bocavirus.
2. primer sets according to claim 1, wherein the primer sets further include drawing shown in SEQ ID NO.17-18
Object.
3. the detection method of seven kinds of diarrhea virus for non-diagnostic purpose, which is characterized in that this method comprises the following steps；
(1) total nucleic acid of sample to be tested is extracted；
(2) reverse transcription is carried out to the total nucleic acid and obtains pcr template；With primer sets of any of claims 1 or 2 to the PCR
Template carries out multiplexed PCR amplification, the material after obtaining multiplexed PCR amplification；
(3) nucleic acid electrophoresis detection is carried out to the material after the multiplexed PCR amplification, obtains the result of nucleic acid electrophoresis detection；
If the amplified production containing 139bp in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection,
It indicates to contain rotavirus A group in the sample to be tested；
If the amplified production containing 364bp in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection,
It indicates to contain norovirus GI type in the sample to be tested；
If the amplified production containing 487bp in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection,
It indicates to contain norovirus GII type in the sample to be tested；
If the amplified production containing 415bp in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection,
It indicates in the sample to be tested containing letter such as virus；
If the amplified production containing 282bp in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection,
It indicates to contain astrovirus in the sample to be tested；
If the amplified production containing 200bp in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection,
It indicates to contain intestinal adenovirus in the sample to be tested；
If the amplified production containing 166bp in the material after the multiplexed PCR amplification described as the result is shown of nucleic acid electrophoresis detection,
It indicates to contain bocavirus in the sample to be tested.
4. detection method according to claim 3, wherein the final of primer shown in SEQ ID NO.3-16 uses concentration
Respectively 0.1-0.8 μM, the final of primer shown in SEQ ID NO.1 using concentration is 0.4-0.8 μM.
5. detection method according to claim 3 or 4, wherein the reaction condition of step (2) includes the program of following a-i:
A:49-51 DEG C, 28-32min；
B:94-96 DEG C, 13-18min；
C:94-96 DEG C, 28-32s,
D:54-56 DEG C, 28-32s,
E:71-73 DEG C, 58-62s, carry out the circulation of 10 c-e；
F:94-96 DEG C, 28-32s,
G:49-51 DEG C, 28-32s,
H:71-73 DEG C, 59-61s, carry out the circulation of 30 f-h；
I:71-73 DEG C, 5-10min.
6. the detection method according to any one of claim 3-5, wherein the nucleic acid electrophoresis detection is solidifying including nucleic acid
Gel electrophoresis and/or nucleic acid Capillary Electrophoresis.
7. the kit that a kind of multiplex PCR detects seven kinds of diarrhea virus, which is characterized in that the kit includes claims 1 or 2
Multiple PCR primer group, reverse transcriptase and the archaeal dna polymerase；Seven kinds of diarrhea virus include rotavirus A group, promise such as
Viral GI type, norovirus GII type, letter such as virus, astrovirus, intestinal adenovirus and bocavirus.
8. kit according to claim 7, which is characterized in that the kit further include PCR reaction buffer and
dNTP。
9. purposes of the primer sets of any of claims 1 or 2 in the kit of preparation seven kinds of diarrhea virus of detection；Wherein, institute
Stating seven kinds of diarrhea virus includes rotavirus A group, norovirus GI type, norovirus GII type, letter such as virus, astrovirus, intestines
Road adenovirus and bocavirus.
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| CN108330211A (en) * | 2017-01-18 | 2018-07-27 | 南京美宁康诚生物科技有限公司 | A group rotavirus/astrovirus/intestinal adenovirus nucleic acid multiple fluorescence PCR detection reagent box and application thereof |
| CN106916906B (en) * | 2017-05-03 | 2020-04-24 | 上海速创诊断产品有限公司 | Primer composition for detecting infectious diarrhea pathogens and kit thereof |
| CN107164562B (en) * | 2017-06-27 | 2021-07-09 | 华东医院 | Diarrhea-associated virus multiple gene detection system and kit and application thereof |
| CN108034762A (en) * | 2017-12-21 | 2018-05-15 | 北京卓诚惠生生物科技股份有限公司 | Multiplex PCR detects six kinds of diarrhea virus primed probe groups |
| CN111748552A (en) * | 2019-12-24 | 2020-10-09 | 深圳市人民医院 | Kit for detecting five infant diarrhea disease series viruses and application thereof |
| CN111826464B (en) * | 2020-07-16 | 2023-08-08 | 亚能生物技术（深圳）有限公司 | Primer probe for detecting various gastrointestinal viruses in one tube, screening method and kit |
| CN112501358B (en) * | 2021-02-04 | 2021-11-09 | 爱科睿特生物医疗科技(南京)有限公司 | Primer probe combination and kit for detecting 9 children digestive tract pathogens |
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