CN103074449A - Kit for synchronously detecting thirteen diarrhea viruses and detection method of kit - Google Patents
Kit for synchronously detecting thirteen diarrhea viruses and detection method of kit Download PDFInfo
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Abstract
The invention discloses a kit for synchronously detecting thirteen diarrhea viruses and a detection method of the kit. The kit comprises DEPC (diethylpyrocarbonate) water, a 5*RT (reverse transcription) buffer, a reverse transcription primer, a reverse transcriptase, an X solution, a 10*PCR (polymerase chain reaction) buffer, a PCR primer, a 25mM magnesium chloride solution, a DNA (deoxyribonucleic acid) polymerase and a positive control, and is characterized in that the reverse transcription primer comprises RT amplification primers of the eleven diarrhea RNA viruses and a human RNA (ribonucleic acid) internal reference, and has a gene sequence shown as SEQ ID NO. 1-12 (sequence identifier number 1-12), and the PCR primer comprises forward and reverse PCR amplification primers of the rest two diarrhea viruses, a human DNA internal reference and a reaction internal reference, and PCR amplification primers of the eleven diarrhea RNA viruses and the human RNA internal reference, and has a gene sequence shown as SEQ ID NO. 13-32. The kit and the detection method have the advantages of high specificity, sensitivity, flux and reliability, low cost, and no false negative results.
Description
Technical field
The present invention relates to detection kit and the detection method thereof of diarrhea virus, especially relate to a kind of test kit and detection method thereof of the 13 kinds of diarrhea viruses of synchronous detection based on GeXP multiple gene expression genetic analysis systems.
Background technology
Diarrhoea is that a kind of sickness rate alimentary infection high and that be popular in all over the world is sick, its sickness rate is only second to upper respiratory tract infection, viral diarrhea is common disease and the frequently-occurring disease of China, cause of disease more complicated, especially larger to infant crowd's harm, therefore need badly and set up diarrhea virus diagnostic method rapidly and efficiently.
At present, the traditional detection method of diarrhea virus mainly comprises following several:
(1) stool for routine inspection: the stool for routine test procedure is to estimate most probable pathogenic agent according to stool proterties, stool examination, age of onset and epidemic season.During the light microscopic microscopy, mucus just, pus and blood stool or bloody stool, can have that volume is red, white corpuscle, be more common in the diarrhoea due to the bacteriums such as salmonella, enteroinvasive E．Coli, enterohemorrhagic Escherichia coli, Campylobacter spp, Yersinia and some virus etc.; Rare just, watery stool, can have a small amount of or without red, white corpuscle, be more common in the diarrhoea due to enterotoxigenic E.Coli, rotavirus, Cryptosporidium, the Aeromonas etc., has cost low, the advantage low to the laboratory hardware requirement, but have following shortcoming: 1) sensitivity is low, false negative often occurs; 2) accuracy is lower, easily causes mistake to examine or mistaken diagnosis.
(2) etiological examination: namely directly do image and examination of living tissue, or the pathogen isolation cultivation, under microscope, Electronic Speculum, observe afterwards, make diagnosis.Advantage: cost is low; Low to the laboratory hardware requirement.Shortcoming: 1) length consuming time: generally need 3-5 days or longer; 2) diagnosis efficiency is low: can only single bacterium pure culture; Pathogenic agent to some phenotypic characteristic variations is difficult to distinguish with the morphology experience; In addition, because antibiotic abuse, bacterium grows in testing process and is suppressed, and causes false-negative result; 3) workload is huge: because every kind of bacterium of each sampling observation sample must be detected, workload is very huge, and particularly part requires comparatively harsh bacterium to culture environment, has more strengthened the workload and the difficulty that detect.
(3) Serological testing: most widely used is Enzyme-multiplied immune technique (ELISA): generally use first primary antibodie and antigen generation specific binding, then with two anti-and primary antibodie generation specific bindings of general enzyme labelling, enzyme is developed the color, afterwards observations again.Advantage: 1) the sample flux is higher: utilize 96 hole enzyme plates, one flat plate can be finished the detection of a plurality of samples; 2) responsive, the characteristic of utilizing enzyme to join can be amplified original antigen signals; 3) quick: as in time, will to shorten much than traditional substratum microorganism culturing test procedure; But also there is following shortcoming: 1) be prone to false positive: owing to wash and antigen coated operation, or cross reaction occurs owing to the antigenic surface determinant is similar, therefore be prone to false positive; 2) take time and effort: making antibody is the work that takes time and effort very much; 3) sensitivity is uncertain: the quality of antibody is depended in the sensitivity of experiment, and the quality of each antibody differs, difficult quality control; 4) can't detect the virus of multi-Vari: variability is virus faster, such as some RNA viruses, has multiple hypotype, and often variation, and variation causes antibody to lose efficacy, can't detectable antigens.
(4) molecular biology---PCR detection method: that uses at present often has real-time fluorescence quantitative PCR, immuno-PCR, a reverse transcription PCR etc., all is that the specific target gene for pathogenic agent detects.Wherein, fluorescent quantitative PCR detection method is the most ripe.The advantage of fluorescent quantitative PCR technique: susceptibility is high, but and accurate quantification, in the detection to Listeria monocytogenes, Salmonellas, Clostridium botulinum, intestinal bacteria etc., all obtained good result.2004, China issued the national standard of implementing fluorescence quantitative PCR detection bird flu H7 hypotype.2009, drugs approved by FDA with the test kit of fluorescence quantitative PCR detection porcine influenza H1N1 hypotype.But also have following shortcoming: 1) flux is low: once can only detect a cause of disease composition, detect multiple pathogenic bacteria such as needs, just need multiple pc R instrument to work simultaneously, efficient is low, and the cycle is long, and the sample of large batch of multiple pathogen contamination is had no way of doing it especially; 2) cost is relatively high: because once detecting a pathogenic agent, when a sample needs to detect multiple pathogenic bacteria simultaneously, must detect one by one, cost increases relatively.
(5) molecular biology---gene chips: gene chip is to pass through micro-processing technology, dna fragmentation (gene probe) with the particular sequence of ten hundreds of and even 1,000,000, arrange regularly and be fixed on the upholders such as silicon chip, slide, a two-dimentional dna probe array that consists of, utilize the biological sample of this class chip and mark to hybridize, can carry out fast qualitative and quantitative analysis to the gene expression profile bioinformation of sample.Advantage: 1) high-flux parallel detects: when a sample needed to detect multiple pathogenic bacteria simultaneously, once experiment can draw whole results; 2) easy and simple to handle quick: whole detection only needed 4-8 hour substantially can go out the result; But also have following shortcoming: 1) technical costs is expensive, complicated: chip of each sample needs, and cost Da Yu $1000/ sample is unfavorable for large-scale promotion; 2) the synthetic and fixing more complicated of probe is particularly made highdensity probe array, is main rate-limiting step; 3) can not accurate quantification, poor repeatability; 4) sensitivity is lower: chip method needs the nucleic acid amount larger, generally must do first the multiplex PCR amplification, because primer is more, easily self produces dimer, hairpin structure, or because the Tm value is different, and the purpose fragment efficient that causes increasing is different, and then the sensitivity of impact detection; 5) because the kind of chip is more, be difficult to formulate a unified quality control standard.
GenomeLab
TMGeXP multiple gene expression genetic analysis systems forms based on capillary electrophoresis separation technology and the research and development of highly sensitive laser Induced Fluorescence Technology of Beckman company maturation, the kapillary display and design in a branch of 8 roads takes full advantage of the alignment characteristics of 96 orifice plates, has reduced cost and the complicacy of using larger display to bring.Adopt multiple PCR method, by Beckman Coulter dye marker, in same EP pipe, analyze simultaneously the expression of a plurality of genes, can fast and effeciently detect the expression situation of gene, overcome the defective of the traditional detection method existence of above-mentioned diarrhea virus, had the following advantages:
1, high-throughput: native system adopts two (96 hole) plates, automatic sample and sample tracer technique, realize a single reaction detection 30-40 site, can do simultaneously 192 reactions (such as 192 patient's samples, 30 kinds of diarrhea viruses of each sample detection, 30 sites), went out the result in one day; For co-infected patients, present method can disposablely provide accurate report, avoids undetected.
2, accuracy is strong: GeXP adopts capillary electrophoresis that the PCR product is carried out separation detection, non-specific amplification product, primer dimer and specific amplification products can be separated, at utmost reduce false positive;
3, susceptibility is high, as a result good reproducibility: the GeXP system has overcome the deviation that the unequal amplification of normal PCR amplification method causes, and has improved a cover goal gene is carried out quantitative speed and susceptibility, adopts laser induced fluorescence(LIF)-PMT, has hypersensitivity;
4, method is easy, and use economical: GeXP provides from a complete set of experimental programs such as reagent, multiple PCR primer design, result and quantitative expression spectrum analysis; The testing cost Shao Yu $50 of each sample is beneficial to large-scale promotion;
5, accurate quantification, handiness are strong: but accurate quantification pathogen gene copy number can be adjusted the target gene of detection at any time according to demand.
6, easily be automated: with regard to sample preparation, Biomek series automated fluid processing instrument can mate fully with GeXP analyser and Ampligrid amplification instrument, and integrated bar code reader has guaranteed that sample is followed the trail of and report the test accurately.
At present, both at home and abroad also not about based on the test kit of 13 kinds of diarrhea viruses of synchronous detection of GeXP multiple gene expression genetic analysis systems and the correlative study report of detection method thereof.
Summary of the invention
Technical problem to be solved by this invention provides a kind of high specificity, highly sensitive, flux is high, reliability is strong, cost is low, without test kit and the detection method thereof based on 13 kinds of diarrhea viruses of synchronous detection of GeXP multiple gene expression genetic analysis systems of false negative result.
The present invention solves the problems of the technologies described above the technical scheme that adopts: the test kit of 13 kinds of diarrhea viruses of a kind of synchronous detection, comprise DEPC water, 5 * RT damping fluid, the reverse transcription primer, ThermoScript II, X solution, 10 * PCR damping fluid, the PCR primer, the 25mM magnesium chloride solution, archaeal dna polymerase and positive reference substance, described reverse transcription primer comprises the RT amplimer of 11 kinds of suffer from diarrhoea RNA viruses and people RNA confidential reference items in the following table 1, described PCR primer comprises remaining two kinds of diarrhoea dna virus in the table 1, people DNA confidential reference items, forward and reverse pcr amplification primer of reaction confidential reference items and the pcr amplification primer of above-mentioned 11 kinds of suffer from diarrhoea RNA viruses and people RNA confidential reference items, gene order is as shown in table 1 below.
Described X solution is for comprising triphosphate deoxy-nucleotide (dNTPs) and universal primer, and described universal primer forward amplimer sequence is AGGTGACACTATAGAATA; Oppositely the amplimer sequence is GTACGACTCACTATAGGGA, described universal primer forward amplimer band fluorescent mark.
Described positive reference substance is the cloning vector of pMD18-T of gene conserved sequence that is connected with the primer of design above-mentioned 13 kinds of diarrhea viruses, RNA confidential reference items, DNA confidential reference items and reaction confidential reference items.
A kind of method of detection diarrhea virus of the test kit that utilizes 13 kinds of diarrhea viruses of synchronous detection specifically may further comprise the steps:
(1) collecting sample and extract nucleic acid
Gather the separation and Culture thing of ight soil, vomitus or the blood of diarrhea patient, from the separation and Culture thing, extract nucleic acid;
(2) carry out the RT reaction take patient's nucleic acid as template
Get the nucleic acid samples RNA5 μ L of 5-20ng/ul, DEPC water 8 μ L, 5 * RT damping fluid, 4 μ L, RT primer solution 2 μ L join on the 96 hole sample panel behind the RT enzyme 1 μ L mixing and carry out reverse transcription, reaction conditions: 48 ° C1 minute; 42 ° C60 minute; 95 ° C5 minute; 4 ° of C are until collect the RT product, each RT primer concentration is 500nM in the wherein reverse transcription primer solution, described RT primer comprises the RT amplimer of 11 kinds of suffer from diarrhoea RNA viruses and people RNA confidential reference items, and gene order is shown in SEQ ID NO.1～NO.12 in the sequence table;
(3) carry out the PCR reaction take reverse transcription product as template
Get RT product 8.6 μ L, 10 * PCR damping fluid, 2 μ L, the magnesium chloride 4 μ L of 25mM, PCR primer solution 2 μ L, archaeal dna polymerase 1.4 μ L join the reaction of the enterprising performing PCR of 96 hole sample panel, reaction conditions behind the X solution 2 μ L mixings: 95 ° C2 minute; 94 ° of C30 seconds, 60 ° of C30 seconds, 70 ° C1 minute, circulate 35 times; 70 ° C1 minute; 4 ° of C are until collect the PCR product; Wherein said X solution is for comprising triphosphate deoxy-nucleotide and universal primer, and described universal primer forward amplimer sequence is AGGTGACACTATAGAATA; Oppositely the amplimer sequence is GTACGACTCACTATAGGGA, described universal primer forward amplimer band fluorescent mark, each PCR primer concentration is 200nM in the PCR primer solution, the PCR primer comprises remaining two kinds of diarrhoea dna virus, people DNA confidential reference items, forward and reverse pcr amplification primer of reaction confidential reference items and the pcr amplification primer of above-mentioned 11 kinds of suffer from diarrhoea RNA viruses and people RNA confidential reference items, and gene order is shown in SEQ ID NO.13～NO.32 in the sequence table;
(4) GeXP genetic analyzer electrocapillary phoresis sample separation
Get PCR product 0.1-1 μ L, the sample-loading buffer 38.75 μ L that the GeXP genetic analyzer is supporting, DNA standard substance 0.5 μ L, join after one in mineral oil mixes on the 96 hole parting liquid plates and carry out the electrocapillary phoresis sample separation, with the collection of illustrative plates and standard diagram contrast that the software of GeXP genetic analyzer obtains, the kind of judgement diarrhea virus.
Compared with prior art, the invention has the advantages that: the present invention discloses test kit and the detection method thereof of 13 kinds of diarrhea viruses of a kind of synchronous detection first, because this test kit has been introduced for people's enteric coronavirus virus, EAd 40,41 types, enterovirus and hypotype thereof (coxsackie virus A 16-type), Astrovirus, rotavirus A, B, the C group, norovirus, the human parechovirus, human bocavirus 2 types, bed ripples virus, little binodal RNA viruses etc., designed specificity amplification primer can detect for 13 kinds of diarrhea viruses simultaneously, can finish the detection of 192 patient's samples within one day, both saved production cost and testing cost, and improved again detection efficiency and shortened the time; The contrast confidential reference items of people DNA and people RNA integrity are guaranteed in checkout procedure false negative is avoided in the judgement of sample quality; Normal reaction contrast confidential reference items (RXN control): monitoring PCR reaction efficiency, avoid false negative, have better sensitivity and specificity so that detect, thereby avoided the not high problem of other detection method specificitys.
In sum, the present invention is based on test kit and the detection method thereof of 13 kinds of diarrhea viruses of synchronous detection of GeXP multiple gene expression genetic analysis systems, can detect for 13 kinds of diarrhea viruses simultaneously, detection sensitivity is high, specificity is good, reduce the false positive rate of conventional pcr amplification, can also effectively solve the easy pollution problem of conventional PCR; Has the Noncompetitive internal comparison system, reliability is strong, without false negative result, utilize GeXP genetic analysis systems sensitivity, accurate quantitative analysis, quick, high-throughout technical superiority, will provide a kind of sensitivity, accurate, quick and multiple gene test scheme cheaply for Disease Control and Prevention Center, hospital and other medical institutions.
Description of drawings
Fig. 1 is the electrocapillary phoresis sample separation result standard collection of illustrative plates of GeXP genetic analyzer.
Embodiment
Embodiment is described in further detail the present invention below in conjunction with accompanying drawing.
Specific embodiment one
The test kit of 13 kinds of diarrhea viruses of a kind of synchronous detection of the present invention, comprise DEPC water, 5 * RT damping fluid, reverse transcription primer (RT primer mix), RT enzyme (full name ThermoScript II), solution X, 10 * PCR damping fluid, the PCR primer, 25mM magnesium chloride solution, archaeal dna polymerase, positive reference substance (the dna fragmentation of particular sequence, be used for the whole reaction system of Quality Control), 5 * RT damping fluid, 10 * PCR damping fluid, 25mM magnesium chloride solution, archaeal dna polymerase are ordered the company in sigma.Above-mentioned reverse transcription primer comprises the RT amplimer of 11 kinds of suffer from diarrhoea RNA viruses and people RNA confidential reference items in the following table 1, above-mentioned PCR primer comprises remaining two kinds of diarrhoea dna virus, people DNA confidential reference items, forward and reverse pcr amplification primer of reaction confidential reference items and the pcr amplification primer of above-mentioned 11 kinds of suffer from diarrhoea RNA viruses and people RNA confidential reference items in the table, and gene order is as shown in table 1 below.(but also shown in the canonical sequence table)
Table 1 diarrhea virus Multiple detection target site and primer nucleic acid sequence table
Above-mentioned X solution comprises triphosphate deoxy-nucleotide (dNTPs) and universal primer, universal primer forward amplimer sequence is AGGTGACACTATAGAATA, oppositely the amplimer sequence is GTACGACTCACTATAGGGA, universal primer forward amplimer band fluorescent mark.
Above-mentioned positive reference substance is the cloning vector of pMD18-T of gene conserved sequence that is connected with the primer of design above-mentioned 13 kinds of diarrhea viruses, RNA confidential reference items, DNA confidential reference items and reaction confidential reference items.
Said gene target site design: the interior conservative gene design primer of special between seed selection, kind
(1) people's enteric coronavirus virus site: can detect everyone enteric coronavirus virus-positive sample
(2) all enterovirus sites: can detect all enterovirus positive
(3) EAd 40,41 type sites: can detect all EAds 40,41 type positive
(4) Astrovirus site: can detect all Astrovirus positive
(5) rotavirus C group site: can detect all rotavirus C group positive
(6) norovirus site: can detect all norovirus positive
(7) human parechovirus site: can detect all human parechovirus's positive
(8) human bocavirus 2 type sites: can detect everyone bocavirus 2 type positive
(9) bed ripples virus site: can detect all bed ripples virus-positive samples
(10) little binodal RNA viruses site: can detect all little binodal RNA viruses positive
(11) rotavirus B group site: can detect all rotavirus B group positive
(12) coxsackie virus A 16-type site: can detect all coxsackie virus A 16-type positive
(13) rotavirus A group site: can detect all rotavirus A group positive
(14) people DNA confidential reference items: select human RNaseP gene, but the integrity of human DNA in the test sample;
(15) people RNA confidential reference items: select human B2M(beta-2-microglobulin) gene, but the integrity of human rna in the test sample;
(16) reaction confidential reference items: select pcDNA3.1, whether monitoring is successfully completed whole reactive system and pcr amplification.
Specific embodiment two
The detection method of 13 kinds of diarrhea viruses of synchronous detection that the present invention is a kind of, the virus that detects comprises people's enteric coronavirus virus, EAd 40,41 types, enterovirus and hypotype thereof (coxsackie virus A 16-type), Astrovirus, rotavirus A, B, the C group, norovirus, the human parechovirus, human bocavirus 2 types, bed ripples virus, (seeing Table 1) such as little binodal RNA viruses, gather patient's sample (stool sample, vomitus, blood etc.), extract nucleic acid, carry out reverse transcription and PCR reaction take patient's nucleic acid as template, finally use electrocapillary phoresis method sample separation, concrete steps are as follows:
1, production is based on the test kit of 13 kinds of diarrhea viruses of synchronous detection of GeXP multiple gene expression genetic analysis systems, and the component that comprises in the test kit is with above-mentioned embodiment 1;
2, collecting sample and extract nucleic acid
Gather the separation and Culture thing of ight soil, vomitus or the blood of diarrhea patient, from the separation and Culture thing, extract nucleic acid;
3, carry out reverse transcription (RT) reaction take patient's nucleic acid as template
1) add reagent and sample (the RT plate sees Table 2) in following ratio in 96 hole sample panel:
Table 2RT reaction reagent and sample mix ratio
| The RT reaction reagent | Amount/hole |
| DEPC(Dnase/Rnase Free) water | 8μL |
| 5 * RT damping fluid | 4μL |
| RT primer (each RT primer concentration is 500nM) | 2μL |
| The RT enzyme | 1μL |
| Sample RNA (5-20ng/ul) | 5μL |
| Total | 20μL |
Annotate: add positive reference substance in the RT reaction, positive reference substance each target pathogenic agent clone gained of serving as reasons comprises the target fragment plasmid, and consumption is 1 μ L/ reaction.
2) hatch (seeing Table 3) by following temperature behind the mixing:
Table 3RT reaction conditions
| The step temperature | Time |
| 148°C | 1 minute |
| 242°C | 60 minutes |
| 395°C | 5 minutes |
| 44°C | Continue: until collect the RT product |
4, carry out the PCR reaction take reverse transcription product as template
1) add reagent and sample (the PCR plate sees Table 4) in following ratio in 96 hole sample panel:
Table 4PCR reaction reagent and sample mix ratio
| The PCR reaction reagent | Amount/ |
| 10 * PCR damping fluid | 2μL |
| 25mM?MgCl2 | 4μL |
| The PCR primer | 2μL |
| Archaeal dna polymerase | 1.4μL |
| X solution | 2μL |
| The RT product | 8.6μL |
| Total | 20μL |
Annotate: X solution comprises triphosphate deoxy-nucleotide (dNTPs) and universal primer, universal primer forward amplimer sequence is AGGTGACACTATAGAATA, oppositely the amplimer sequence is GTACGACTCACTATAGGGA, universal primer forward amplimer band fluorescent mark.
2) carry out thermal cycle reaction (seeing Table 5) by following temperature behind the mixing:
Table 5PCR reaction conditions
| Step | Temperature | Time |
| 1 | 95°C | 2 minutes |
| 2 | 94°C | 30 seconds |
| 3 | 60°C | 30 seconds |
| 4 | 70°C | 1 minute |
| 5 | N/A | Repeat 2-4 step 34 time (totally 35 times) |
| 6 | 70°C | 1 minute |
| 7 | 4°C | Continue: until collect the PCR product |
5, GeXP genetic analyzer electrocapillary phoresis sample separation
1) preparation GeXP sample (seeing Table 5):
Table 5GeXP sample mix ratio
| The GeXP sample | Amount/hole |
| Sample-loading buffer (Beckman Gexp system support) | 38.75μL |
| DNA size criteria 400 | 0.5μL |
| The PCR product | 0.1-1μL |
| Mineral oil | 1 |
2) electrocapillary phoresis sample separation
The GeXP sample is added in the hole of proper number on the 96 hole capillary electrophoresis separation plates and carry out capillary electrophoresis separation; Capillary electrophoresis separation is the Novel liquid-phase isolation technique of a class take kapillary as split tunnel, take high-voltage dc as motivating force, and specific procedure is 90 ℃ of sex change 120 seconds, sample introduction voltage 2kv, 30 seconds, separation voltage 6kv, 35 minutes.
6, interpretation of result (seeing GeNO.meLab GeXP genetic analyzer specification sheets)
According to the parameter of giving tacit consent on the own software of GeXP genetic analyzer the result is carried out the clip size analysis, its X-coordinate represents clip size, and ordinate zou is that signal is strong and weak.With the collection of illustrative plates and standard diagram contrast that the software of GeXP genetic analyzer obtains, the kind of judgement diarrhea virus.Standard diagram as shown in Figure 1, its result can accurately detect 13 kinds of target diarrhea viruses, each target fragment size interval is moderate, and signal is unlikely to supersaturation, signal is relatively fair between each target, and does not have broad peak, the phenomenon such as bimodal.
Specific embodiment three
Detection kit sensitivity, specificity analyses
Sensitivity analysis: positive control by behind certain copy number doubling dilution, is detected through pcr amplification and capillary electrophoresis until can't detect signal, and this copy number is the lowest detection line, namely the sensitivity of test kit.Sensitivity reaches 40 copy/uL.
Specificity analyses: it is the unimodal of target fragment size that the substance pcr amplification detects through capillary electrophoresis.
Above-mentioned explanation is not limitation of the present invention, and the present invention also is not limited to above-mentioned giving an example.Those skilled in the art are in essential scope of the present invention, and the variation of making, remodeling, interpolation or replacement also should belong to protection scope of the present invention.
Claims (4)
1. the test kit of 13 kinds of diarrhea viruses of a synchronous detection, comprise DEPC water, 5 * RT damping fluid, the reverse transcription primer, ThermoScript II, X solution, 10 * PCR damping fluid, the PCR primer, the 25mM magnesium chloride solution, archaeal dna polymerase and positive reference substance, it is characterized in that described reverse transcription primer comprises the RT amplimer of 11 kinds of suffer from diarrhoea RNA viruses and people RNA confidential reference items in the following table, described PCR primer comprises remaining two kinds of diarrhoea dna virus in the table, people DNA confidential reference items, forward and reverse pcr amplification primer of reaction confidential reference items and the pcr amplification primer of above-mentioned 11 kinds of suffer from diarrhoea RNA viruses and people RNA confidential reference items, gene order is as shown in the table.
2. the test kit of 13 kinds of diarrhea viruses of a kind of synchronous detection according to claim 1, it is characterized in that: described X solution is for comprising triphosphate deoxy-nucleotide (dNTPs) and universal primer, and described universal primer forward amplimer sequence is AGGTGACACTATAGAATA; Oppositely the amplimer sequence is GTACGACTCACTATAGGGA, described universal primer forward amplimer band fluorescent mark.
3. the test kit of 13 kinds of diarrhea viruses of a kind of synchronous detection according to claim 1 is characterized in that: described positive reference substance is the cloning vector of pMD18-T of gene conserved sequence that is connected with the primer of the above-mentioned 13 kinds of diarrhea viruses of design, RNA confidential reference items, DNA confidential reference items and reaction confidential reference items.
4. the method for the detection diarrhea virus of a test kit that utilizes 13 kinds of diarrhea viruses of synchronous detection claimed in claim 1 is characterized in that specifically may further comprise the steps:
(1) collecting sample and extract nucleic acid
Gather the separation and Culture thing of ight soil, vomitus or the blood of diarrhea patient, from the separation and Culture thing, extract nucleic acid;
(2) carry out the RT reaction take patient's nucleic acid as template
Get the nucleic acid samples RNA5 μ L of 5-20ng/ul, DEPC water 8 μ L, 5 * RT damping fluid, 4 μ L, RT primer solution 2 μ L join on the 96 hole sample panel behind the RT enzyme 1 μ L mixing and carry out reverse transcription, reaction conditions: 48 ° C1 minute; 42 ° C60 minute; 95 ° C5 minute; 4 ° of C are until collect the RT product, each RT primer concentration is 500nM in the wherein reverse transcription primer solution, described RT primer comprises the RT amplimer of 11 kinds of suffer from diarrhoea RNA viruses and people RNA confidential reference items, and gene order is shown in SEQ ID NO.1～NO.12 in the sequence table;
(3) carry out the PCR reaction take reverse transcription product as template
Get RT product 8.6 μ L, 10 * PCR damping fluid, 2 μ L, the magnesium chloride 4 μ L of 25mM, PCR primer solution 2 μ L, archaeal dna polymerase 1.4 μ L join the reaction of the enterprising performing PCR of 96 hole sample panel, reaction conditions behind the X solution 2 μ L mixings: 95 ° C2 minute; 94 ° of C30 seconds, 60 ° of C30 seconds, 70 ° C1 minute, circulate 35 times; 70 ° C1 minute; 4 ° of C are until collect the PCR product; Wherein said X solution is for comprising triphosphate deoxy-nucleotide and universal primer, and described universal primer forward amplimer sequence is AGGTGACACTATAGAATA; Oppositely the amplimer sequence is GTACGACTCACTATAGGGA, described universal primer forward amplimer band fluorescent mark, each PCR primer concentration is 200nM in the PCR primer solution, the PCR primer comprises remaining two kinds of diarrhoea dna virus, people DNA confidential reference items, forward and reverse pcr amplification primer of reaction confidential reference items and the pcr amplification primer of above-mentioned 11 kinds of suffer from diarrhoea RNA viruses and people RNA confidential reference items, and gene order is shown in SEQ ID NO.13～NO.32 in the sequence table;
(4) GeXP genetic analyzer electrocapillary phoresis sample separation
Get PCR product 0.1-1 μ L, the sample-loading buffer 38.75 μ L that the GeXP genetic analyzer is supporting, DNA standard substance 0.5 μ L, join after one in mineral oil mixes on the 96 hole parting liquid plates and carry out the electrocapillary phoresis sample separation, with the collection of illustrative plates and standard diagram contrast that the software of GeXP genetic analyzer obtains, the kind of judgement diarrhea virus.
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| CN103981286A (en) * | 2014-05-19 | 2014-08-13 | 广西壮族自治区兽医研究所 | GeXP rapid detection kit for identifying eight types of porcine viral diseases and primer group thereof |
| CN103981286B (en) * | 2014-05-19 | 2016-08-24 | 广西壮族自治区兽医研究所 | Differentiate GeXP rapid detection kit and the primer sets thereof of 8 kinds of virus diseases of pigs |
| CN104531899A (en) * | 2014-12-26 | 2015-04-22 | 广西壮族自治区兽医研究所 | GeXP quick detection kit for avian influenza virus and H6 subtype and N1 subtype thereof |
| CN105316430A (en) * | 2015-11-27 | 2016-02-10 | 广西壮族自治区兽医研究所 | GeXP rapid detection primer group and kit for identifying H5N1 and H9N2 subtype avian influenza viruses synchronously and application of primer group and kit |
| CN105316430B (en) * | 2015-11-27 | 2018-09-25 | 广西壮族自治区兽医研究所 | Differentiate the quick detection primer groups of the GeXP of H5N1 and H9N2 subtype avian influenza virus, kit and its application simultaneously |
| 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 |
| 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 |
| CN111269995A (en) * | 2018-12-04 | 2020-06-12 | 深圳华大因源医药科技有限公司 | Primer group, kit and detection method for detecting pathogen |
| CN111269995B (en) * | 2018-12-04 | 2023-12-26 | 深圳华大因源医药科技有限公司 | Primer group, kit and detection method for detecting pathogen |
| CN112725537A (en) * | 2020-03-12 | 2021-04-30 | 宁波海尔施基因科技有限公司 | Multiplex real-time fluorescent quantitative PCR (polymerase chain reaction) kit and method for detecting 2019 novel coronavirus and primer probe composition |
| CN114736991A (en) * | 2022-05-18 | 2022-07-12 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting diarrheagenic virus by one-step method and application of composition, kit and method |
| CN114736991B (en) * | 2022-05-18 | 2023-11-17 | 圣湘生物科技股份有限公司 | Composition, kit, method and application for detecting diarrheal virus by one-step method |
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