CN103146846A

CN103146846A - Single standard product-based four-color fluorogenic quantitative PCR (Polymerase Chain Reaction) method and kit

Info

Publication number: CN103146846A
Application number: CN2013100920748A
Authority: CN
Inventors: 刘健翊; 周孟; 陈瑶; 王宝玉; 廖杰; 叶奕东; 黄佳柳; 莫海月; 徐德峰; 黄常艳; 徐贵云
Original assignee: GUANGZHOU VIPOTION BIOTECHNOLOGY CO Ltd
Current assignee: GUANGZHOU VIPOTION BIOTECHNOLOGY CO Ltd
Priority date: 2013-03-21
Filing date: 2013-03-21
Publication date: 2013-06-12

Abstract

The invention discloses a single standard product-based quardruple four-color fluorogenic quantitative PCR (Polymerase Chain Reaction) technology. The technology is capable of detecting group A rotavirus (HRVA), norovirus group I (NVGI), norovirus group II (NVG II) and human astrovirus (HAstV) RNA in various treated specimen through one-step quardruple PCR amplification and quantifying virus load. The technology is characterized in that single standard product is adopted as positive control and quantitative standard in the quardruple four-color fluorogenic quantitative PCR detection for four viruses, thereby breaking through the deficiency that the traditional multiple fluorogenic quantitative PCR needs a plurality of standard products, improving the experiment detection efficiency, reducing the pollution probability and improving the preparation and production efficiency of the kit. The method and kit can be used for fast high-throughput test and epidemic monitoring in laboratories of HRVA, NVG I, NVG II and HAstV which can cause diarrhea syndromes. The single standard product-based four-color florogenic quantitative PCR method and the kit belong to the biotechnology field.

Description

A kind of four look fluorescence quantifying PCR method and test kits based on the single standard product

Technical field

The present invention relates to viral nucleic acid and detect, belong to biological technical field.The present invention adopts the fluorescent quantitative PCR technique based on quadruple four looks of single standard product, by A group rotavirus (HRVA), norovirus I group (NVG I), norovirus II group (NVG II) and people's Astrovirus (HAstV) RNA in sample after all kinds of processing of single stage method pcr amplification detection, for laboratory rapid detection and the epidemic situation monitoring of the rotavirus, norovirus I group, norovirus II group and the people's Astrovirus that cause the diarrhoea syndrome.The present invention also has higher requirement to the design of primer and probe and the enforcement of experiment.

Background technology

WHO estimates that the whole world annual diarrhoea case is inferior up to 30-50 hundred million examples, has 50-100 ten thousand cases dead because of severe diarrhea, and average every day, dead 2.5 ten thousand people, more outstanding children especially.The various acute gastroenteritiss of children's are more common clinically, can cause the symptoms such as the patient vomits, stomachache, diarrhoea, are a kind of acute infectious diseases, and general onset is more anxious, and heating and general malaise symptom can be arranged.The cause of disease that causes acute diarrhea is a lot, can comprise bacteriophage or other microorganisms.After finding norwalk virus in 1972, in succession find and established and multiplely cause the virus of diarrhoea and can directly detect virus in the diarrhea patient stool.What research was more at present is that various pathogenic virus comprise rotavirus (Rotavirus, RV), norovirus (Norovirus, NV), people's Astrovirus (Human Astrovirus, HAstV) and enterovirns type 71 (Enterovirus 71, EV71), Coxsackie virus (Coxsackie virus, CAV), letter is as virus (Sapovirus, SV) and enteric adenovirus (Enteric adenovirus, EADV) etc.

Rotavirus main infection infant, autumn and winter is occurred frequently; EAd main infection infant below 2 years old, summer and autumn, sickness rate was higher.Rotavirus belongs to the Reoviridae rotavirus, and virus particle is rounded, and by likeness in form wheel and gaining the name, for without togavirus, virogene contains the bifilar line style RNA of 11 sections.At present humans and animals shape virus is divided into A～D4 group.The A group is Ordinary Rotavirus (mainly causing infantile diarrhea); The B group is porcine rotavirus and adult diarrhea rotavirus (Adult diarrhea rotavirus), and the C group is people and porcine rotavirus; The D group is chicken and birds rotavirus.Have 6 structural protein of AMPLIGEN genome encoding (VP1-4, VP6-7) and five Nonstructural Proteins (NSP1-5) of 11 segments, be wrapped in three layers of protein coat; Skin is comprised of two albumen, VP4 and VP7, VP4 (relative molecular mass is 88000) albumen is the surface that the spike furcella protrudes from virus particle, VP5 comprises host cell corresponding circle of sensation amino acid 384-401, with potential corresponding circle of sensation sequence and antigen around amino acid 305 relevant.VP7 is the main capsid protein of virus.The present invention detects mainly for the A group rotavirus.

Norovirus is the pathogenic agent of separating patient's ight soil of an acute diarrhea breaking out in U.S.'s Cécile Nowak city from nineteen sixty-eight the earliest, is the sub-thread positive chain RNA, and total length is 7642nt approximately, comprises 3 open reading frame (Open Reading Frame, ORF).Its more than 100 strain isolateds are carried out gene sequencing at present.Different areas, the world, the NV gene RNA polymerase region sequence that different time is popular are relatively conservative, and the nucleotide amino acid sequence homology is higher than 60%, and what have reaches more than 90%.NV comprises 5 gene groups, and that infects the people comprises gene I, II and IV group, and wherein the most common with the II group, I group is accidental, the rare report of IV.Gene group I (Genogroup I, NVG I) also comprises Southampton's virus (Southampton virus), sandstorm virus (Desert Shield virus); Gene group II (Genogroup II, NVG II) also comprises snow mountain virus (Snow Mountain virus), hawaii virus (Hawaii virus), Toronto virus (Toronto virus) etc.Therefore at present the detection primer of norovirus take for I (NVG I) and II (NVG II) group two kinds of genotype as main.Norovirus and the Sapporo sample virus (Sapporo-like Virus, SLV) of finding in Japan, present formal name is called letter as virus (Sapovirus, SV), is collectively referred to as mankind's Calicivirus.

Astrovirus gastro-enteritis mainly occurs in children below 5 years old, also is found in the elderly.Astrovirus was adopted in the stool sample of Electronic Speculum at diarrhea children by Appleton and Higgins in 1975 and finds first, Astroviridae comprises mammals Astrovirus (Mamastroviruses) and two genus of bird Astrovirus (Avastroviruses), and human astrovirus virus belongs to the mammals Astrovirus.Astrovirus is spherical in shape, and nucleocapsid is 20 bodies of rule, and without after birth, the virion diameter is 28nm, and approximately 10% virion has distinctive 5～6 angles; The virion diameter that cell cultures obtains is 41nm, comprises the furcella of 10nm.It is popular serotype the most widely that human astrovirus virus can be divided into 7 serotypes (AstV21～AstV27), 7 genotype, human astrovirus virus serotype 1(HastV21).Nucleic acid is single positive chain RNA, 7.0kb, and two ends are non-coding region, there are three overlapping open reading frames (ORF) centre.This virus is worldwide distribution.

In recent years, along with the national health competent authorities pay attention to day by day to viral diarrhea clinical diagnosis and epidemic prevention and control, the laboratory of the pathogenic agent of viral diarrhea syndrome such as A group rotavirus (HRVA), norovirus I group (NVG I), norovirus II group (NVG II) and people's Astrovirus (HAstV) etc. is detected becomes an extremely important job.

Above-mentioned various virus serotype is numerous, and the syndrome of suffering from diarrhoea is clinically caused jointly by one or more viruses often, and this is the universal phenomenon of viral infection diarrhoea.In laboratory diagnostic method, traditional virus culture and serum neutralization test method are important evidence, but have time-consuming, loaded down with trivial details and the not high limitation of susceptibility, and the diversity of serotype limits the application of its Serology test.The diagnosis that develops into enterovirus and the classification of molecular Biological Detection technology provide fast method accurately.

RNA viruses diagnostic techniques based on the RT-PCR method is the highest RNA detection technique of present sensitivity.It becomes article one chain of complementary DNA (cDNA) take RNA as the template reverse transcription, carry out again the PCR reaction take this chain as template, can detect thus the specific detection of utmost point low copy number viral nucleic acid in sample, overcome that traditional technique in measuring sensitivity is low, the shortcoming of complex operation.The method accurate quick generally can obtain detected result accurately in 3-4 hour, be present comparatively ideal diagnostic techniques, was widely applied in medical science and field of biology.The method mainly depends on for specific primers of design such as A group rotavirus (HRVA), norovirus I group (NVG I), norovirus II group (NVG II) and people's Astroviruss (HAstV), and detects after sample is increased.

Development along with round pcr, fluorescent quantitative PCR technique take specificity T aqman fluorescent probe as characteristics, the operation of Implementing Complete stopped pipe type, can not only greatly reduce the chance that amplified production pollutes, and than the conventional RT-PCR technology, Sensitivity Specificity all has more clear superiority, compares through the detection of clinical sample, the method has high specificity, can detect 10 ²The viral nucleic acid copy number of the order of magnitude, and more responsive, quick and easy than conventional RT-PCR and isolation of virus.Common RT-PCR is from viral nucleic acid extraction, RT-PCR reaction and electrophoresis, whole process approximately needed about 3-4 hour, and adopt fluorescent quantitative PCR technique from nucleic acid extraction to completing detection, only need approximately 2 hours, and can complete simultaneously the detection to the same viral nucleic acid of a plurality of pattern detection.

Multiple fluorescence quantitative PCR has overcome the single kind of shortcoming that viral single-gene detects, and can detect for the Various Diseases virus polygene simultaneously, has truly realized high throughput testing.Detect after all kinds of processing A group rotavirus (HRVA), norovirus I group (NVG I), norovirus II group (NVG II) and people's Astrovirus (HAstV) RNA in sample by the single stage method pcr amplification in same PCR reaction tubes, and use different fluorescent quantitation standard substance, carry out the monitoring of pcr amplification and viral load quantitatively, be used for causing laboratory rapid detection and the epidemic situation monitoring of A group rotavirus, norovirus I group, norovirus II group and people's Astrovirus of diarrhoea syndrome.

method of the present invention is the fluorescent quantitative PCR technique based on quadruple four looks of single fluorescent quantitation standard substance, if consistent according to gene amplification efficient, the mathematical principle of the initial copy number of template that so identical CT(Threshold Cycle) value is corresponding identical, for A group rotavirus (HRVA), norovirus I group (NVG I), norovirus II group (NVG II) and the same amplification standard substance of four kinds of virus formulations of people's Astrovirus (HAstV), detect the A group rotavirus by the single stage method pcr amplification in same PCR reaction tubes, norovirus I group, existence and the viral load of norovirus II group and people's Astrovirus RNA.

The present invention also has higher requirement to the design of primer and probe and the enforcement of experiment.

Innovation of the present invention be to break through traditional multiple fluorescence quantitative PCR need to prepare the multiple standards product deficiency, improved the technique of reagent preparation and the efficient of detection.

The invention further relates to a kind of A group rotavirus (HRVA) based on the single standard product, norovirus I group (NVG I), norovirus II group (NVG II) and people's Astrovirus (HAstV) quadruple four look fluorescent quantificationally PCR detecting kits.

Summary of the invention

method of the present invention is the fluorescent quantitative PCR technique based on quadruple four looks of single fluorescent quantitation standard substance, under the consistent condition of gene amplification efficient, identical CT(Threshold Cycle) the initial copy number of template corresponding to value, for the A group rotavirus, norovirus I group, four kinds of virus specific primers of design of norovirus II group and people's Astrovirus and probe, adopt FAM, HEX, four kinds of fluorescent signal passages of ROX and CY5, optimizing reaction system and reaction conditions, detect above-mentioned viral RNA by the single stage method pcr amplification in same PCR reaction tubes.

Detection method experimental procedure of the present invention comprises:

1) infect or doubtful sample collection and transporting;

2) sample preprocessing and extraction RNA;

3) single stage method quadruple four look quantitative fluorescent PCRs detect sample;

4) carry out accurate quantification according to the virus in the amplification curve sample of single standard product.

According to above-mentioned technological breakthrough, a kind of A group rotavirus based on the single standard product, norovirus I group, norovirus II group and people's Astrovirus quadruple four look fluorescent quantificationally PCR detecting kits have been the present invention further provides.

According to a preferred embodiment of the invention, the present invention has higher requirement to the design of primer and probe.

According to a preferred embodiment of the invention, principle of design and requirement according to primer and the probe of multiple fluorescence quantitative PCR, A group rotavirus (HRVA), norovirus I group (NVG I), norovirus II group (NVG II) and four kinds of viruses of people's Astrovirus (HAstV) are carried out sufficient bioinformatic analysis, and primer specific according to conserved regions design and fluorescent probe are as follows:

The A group rotavirus:

Upstream primer HRVA-F1:5 '-TGACCCTCTATGAGCACAATAGTT-3 ' (SEQ ID NO.1);

Downstream primer HRVA-R1:5 '-CACATAACGCCCCTATAGCC-3 ' (SEQ ID NO.2);

Fluorescent probe HRVA-P:

5’-FAM-AAAGCTAACACTGTCAAAAACCTAA-BHQ1-3’（SEQ ID NO.3）；

Norovirus I group:

Upstream primer NVG I-F1:5 '-TGGATGCGBTTCCATGA-3 ' (SEQ ID NO.4);

Downstream primer NVG I-R1:5 '-CCTTAGACGCCATCATCATTTAC-3 ' (SEQ ID NO.5);

Fluorescent probe NVG I-P:

5’-HEX-CAGGAGATCGCAATCTCCTGCCCGA-BHQ1-3’（SEQ ID NO.6）；

Norovirus II group:

Upstream primer NVG II-F1:5 '-ATTCTCDGAYCTSAGCACRT-3 ' (SEQ ID NO.7);

Downstream primer NVG II-R1:5 '-TCGACGCCATCTTCATTCAC-3 ' (SEQ ID NO.8);

Fluorescent probe NVG II-P:5 '-ROX-AGGGCGATCGCAATCTGGCTCCC-BHQ1-3 ' (SEQ ID NO.9);

People's Astrovirus:

Upstream primer HAstV-F1:5 '-CATCTGGAAGCCGCGG-3 ' (SEQ ID NO.10);

Downstream primer HAstV-R1:5 '-TAAAATAATCTAAACAGAGACAGAAAAG-3 ' (SEQ ID NO.11);

Fluorescent probe HAstV-P:5 '-CY5CACGCCGAGTAGGAACGAGGGTACAG-BHQ1-3 ' (SEQ ID NO.12);

According to a preferred embodiment of the invention, wherein said detection to the specific target zone of A group rotavirus (HRVA), norovirus I group (NVG I), norovirus II group (NVG II) and four kinds of viral RNAs of people's Astrovirus (HAstV) is:

The A group rotavirus:

TGACCCTCTATGAGCACAATAGTTAAAAGCTAACACTGTCAAAAACCTAAATGGCTATAGGGGCGTTATGTG（SEQ ID NO.13）；

Norovirus I group:

TGGATGCGCTTCCATGATCTCGGATTGTGGACAGGAGATCGCGATCTCCTGCCCGAATTCGTAAATGATGATGCCGTCTAAGG（SEQ ID NO.14）；

Norovirus II group:

ATTCTCGGACCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGTTTTGTGAATGAAGATGGCGTCGA（SEQ ID NO.15）；

People's Astrovirus:

CATCTGGAAGCCGCGGCCACGCCGAGTAGGAACGAGGGTACAGCTTCCTTCTTTTCTGTCTCTGTTTAGATTATTTTA（SEQ ID NO.16）。

The present invention is the quadruple four look fluorescence quantifying PCR methods of single standard product, according to a preferred embodiment of the invention, analyze and introduce the nonsense sequence for the target sequence of above-mentioned A group rotavirus (HRVA), norovirus I group (NVG I), norovirus II group (NVG II) and four kinds of viruses of people's Astrovirus (HAstV) between each sequence, design 4 sections different standard substance sequences, each fragment is between 100-120bp, the joint has the 6-8 of being no less than sequence to repeat, and increases for the second time take viral target fragment as masterplate.Mix as template in the external fragment that all are corresponding and be the sliceable single Long fragment gene that obtains through pcr amplification for the third time, as the nucleotide sequence of single standard product, be cloned into the pMD18-T carrier, be prepared into standard substance.

According to a preferred embodiment of the invention, wherein said test kit is a kind of test kit that detects A group rotavirus (HRVA), norovirus I group (NVG I), norovirus II group (NVG II) and four kinds of viruses of people's Astrovirus (HAstV) for quick real-time quantitative, and this test kit comprises:

1) viral nucleic acid extracts reagent;

2) reversed transcriptive enzyme system and Taq enzyme are;

3) four look Fluorescence PCR systems (PCR MIX);

4) single positive standard substance and negative quality control product etc.

according to a preferred embodiment of the invention, wherein the four quantitative fluorescent PCR reaction systems (PCR MIX) of the above-mentioned four kinds of viral RNAs of said detection are by A group rotavirus (HRVA) specificity upstream and downstream primer, , a specificity fluorescent probe (FAM fluorescent mark), norovirus I group's (NVG I) specificity upstream and downstream primer, , a specificity fluorescent probe (HEX fluorescent mark), norovirus II group (NVG II) specificity upstream and downstream primer, , a specificity fluorescent probe (ROX fluorescent mark), the specificity upstream and downstream primer of people's Astrovirus (HAstV), , a specificity fluorescent probe (CY5 fluorescent mark), PCR Buffer(includes magnesium ion, Tris-HCl etc.), four kinds of nucleotide monomers (dNTPs), the reaction system that the compositions such as pcr amplification toughener and deionized water consist of.

According to a preferred embodiment of the invention, the wherein preparation in the following manner of said PCR reaction system (PCR MIX):

Every part of reaction solution (PCR MIX) volume is 20.0ul, and the preparation method is as follows:

10×Buffer 2.5ul

dNTP（10mM） 0.75ul

HRVA-F1（50uM） 0.2ul

HRVA-R1（50uM） 0.2ul

NVGⅠ-F1（50uM） 0.2ul

NVGⅠ-R1（50uM） 0.2ul

NVGⅡ-F1（50uM） 0.2ul

NVGⅡ-R1（50uM） 0.2ul

HAstV-F1（50uM） 0.2ul

HAstV-R1（50uM） 0.2ul

HRVA-P（50uM） 0.15ul

NVGⅠ-P（50uM） 0.15ul

NVGⅡ-P（50uM） 0.15ul

HAstV-P（50uM） 0.15ul

NH4Cl（0.5M） 0.5ul

DDH2O 14.55ul

Amount to 20.0ul

Add according to quantity successively following reagent during use,

Reversed transcriptive enzyme system	1.0ul
		Taq enzyme system	1.0ul
RNA/ standard substance/negative control	3.0ul

Be totally 25ul, then carry out carrying out simultaneously A group rotavirus (HRVA), norovirus I group (NVG I), norovirus II group (NVG II) and four kinds of Detectings of people's Astrovirus (HAstV) in a PCR pipe.

Need to be placed on-20 ℃ of preservations after the preparation of above system, wherein PCR buffer is by 100mM Tris-HCl(pH8.0), 50mM MgCl ₂, the composition such as 500mM KCl; The H that the preparation mentioned reagent is used ₂O is and can uses after DEPC processes water process autoclaving; Reaction system adds NH ₄Cl(0.5M) work and be 10mM NH ⁴⁺Can strengthen in quadruple quantitative fluorescent PCR reaction the specificity that primer probe separately is combined with corresponding template, avoid the cross reactivity between the primer probe.

according to a preferred embodiment of the invention, wherein said standard substance are for to A group rotavirus (HRVA), norovirus I group (NVG I), the copy number of norovirus II group (NVG II) and four kinds of viruses of people's Astrovirus (HAstV) carries out the single fluorescent quantitation standard substance of accurate quantification, increase for the second time after external target fragment adjustment with four kinds of virus correspondences, and the product mixing is obtained single Long fragment gene as template through pcr amplification splicing for the third time, according to this as the nucleotide sequence of single standard product, be cloned into the pMD18-T carrier, the single standard product that are prepared into, need not to prepare four kinds of standard substance.

According to a preferred embodiment of the invention, wherein its accurate copy number of said standard substance is 10 ⁷Copies/ml, negative quality control product process and autoclaved TE damping fluid through DEPC.

According to a preferred embodiment of the invention, said standard substance (10 wherein ⁷Copies/ml), successively according to 1.0 * 10 ⁶, 1.0 * 10 ⁵, 1.0 * 10 ⁴, 1.0 * 10 ³, 1.0 * 10 ², 1.0 * 10 ¹Copies/ml carries out sensitivity experiment, and proved test kit detection sensitivity is as a result: 1.0 * 10 ²Copies/ml, the susceptibility of this quadruple fluorescence quantifying PCR method and accuracy are higher than the method for the RT-PCR electrophoresis detection of routine.Negative quality control product is to process and autoclaved TE damping fluid through DEPC.

On the other hand, according to a preferred embodiment of the invention, test kit is furnished with viral nucleic acid and extracts reagent, it is the RNA extracting solution, mainly contain 4M Guanidinium hydrochloride, 0.1MNaAc, 0.015M Trisodium Citrate and 0.4% β mercaptoethanol etc., the reagent such as preparing voluntarily chloroform, Virahol of in addition still needing is used for extracting viral RNA.

According to a preferred embodiment of the invention, the method of the detection A group rotavirus (HRVA) of inventing, norovirus I group (NVG I), norovirus II group (NVG II) and four kinds of viral RNAs of people's Astrovirus (HAstV) is single stage method, namely reverse transcription reaction be need not carry out separately, can reverse transcription and detection by quantitative be realized in the quantitative real time PCR Instrument previous step.

The fluorescent quantitative PCR condition that wherein adopts is: the Mx3000P of Stratagene company and Mx3005P etc., ABI PRISM7300/7500, ABI PRISM 7700, ABI PRISM5700, ABI GeneAmp 7000, MJ Opticon etc. use quantitative real time PCR Instrument, cycling condition is 42 ℃ → 20 minutes, then 93 ℃ → 2 minutes, then 93 ℃ 30 seconds → 55 ℃ 45 seconds (gathering fluorescent signal in this step), 40 circulations; The fluorescence such as LightCycler are decided the quantitative PCR instrument, and cycling condition is 42 ℃ → 20 minutes, and 93 ℃ → 2 minutes, then 93 ℃ 5 seconds → 55 ℃ gathered fluorescent signal, totally 40 circulations in this step in 45 seconds.

According to a preferred embodiment of the invention, after reaction finishes, use the manual setting threshold value to make negative quality control product Ct value more than 40, the Ct value is positive less than 35 circulations; The Ct value is gray area between 35-40 circulation, need to carry out revision test.After revision test, if the Ct value still 35-40 the circulation between, be judged as the positive, do not have fluorescent signal negative.

Sensitivity experiment: according to a preferred embodiment of the invention, with 1 * TEbuffer(10mM Tris-HCl, 1mM EDTA, pH=8.0) dilution standard product (10 ⁷Copies/ml), respectively as linearity and sensitivity reference material, successively according to 1.0 * 10 ⁶, 1.0 * 10 ⁵, 1.0 * 10 ⁴, 1.0 * 10 ³, 1.0 * 10 ², 1.0 * 10 ¹Copies/ml, each extent of dilution is made 3 parts of replications.Analyze | the r| value all can reach | and r| 〉=0.99, the sensitivity of this test kit are 1.0 * 10 ²Copies/ml.

Repeated experiment: according to a preferred embodiment of the invention, the detection by quantitative accuracy determination method of test kit is as follows: detect on same pcr amplification instrument with three batches of these test kits of quantity-produced and observe it | the variation of r| value and detection sensitivity.Experiment conclusion is: | r| 〉=0.97, quantitative precision CV＜10%, as shown in table 1:

The official written reply of table 1. test kit repeated experiment and variation within batch coefficient

Standard substance/ml-1 criticizes interior CV(%) batch between CV(%)

10 ⁷3.4 5.1

10 ⁶3.5 4.3

10 ⁵3.5 4.2

10 ⁴4.9 3.8

10 ³5.1 3.2

10 ²5.5 4.7

specificity experiment: according to a preferred embodiment of the invention, the detection by quantitative specific assay method of test kit is as follows: with this test kit to A group rotavirus (HRVA), norovirus I group (NVG I), the sample of norovirus II group (NVG II) and four kinds of virus infectiones of people's Astrovirus (HAstV) (amounting to 500 examples) and ' negative ' specimens detect, wherein detect A group rotavirus (HRVA) 78 examples, norovirus I group (NVG I) 1 example, norovirus II group (NVG II) 2 example and four kinds of viruses of people's Astrovirus (HAstV) 1 example, result is consistent with the detected result of the test method that the Ministry of Health five large syndrome monitoring scheme provides, identify by order-checking and confirm, True Positive Rate and true negative rate are 100%.

Stability test: test kit of the present invention confirms to have good repeatability and stable through repeatedly different repeated experiments checkings.The test kit validity period can reach 10 months under-20 ℃ of conditions.

The fluorescent quantitative PCR technique based on quadruple four looks of single standard product that the present invention sets up, method by A group rotavirus (HRVA), norovirus I group (NVG I), norovirus II group (NVG II) and people's Astrovirus (HAstV) RNA in sample after all kinds of processing of single stage method pcr amplification detection, Quadruple-PCR primer and the four look fluorescent probes designed to above-mentioned four kinds of nucleic acid sequences, realized that to sample reaction detects the purpose of four kinds of viruses simultaneously, it is very convenient to use.Judgement from the processing sample to result only needed about 2 hours.So compare simple to operate, advantage quickly and easily with traditional method.

Therefore, test kit of the present invention provides new high-throughout detection method for rapid detection and the large sample generaI investigation of clinical samples.

On the other hand, the present invention adopts the single standard product as positive control and the quantitative criterion of experiment, and the above-mentioned viral RNA that infects is carried out rapid detection and quantitative, makes detection more efficient and convenient, has improved the efficient that experiment detects, and has reduced the chance of polluting.Laboratory rapid detection and epidemic situation monitoring for the rotavirus, norovirus I group, norovirus II group and the people's Astrovirus that cause the diarrhoea syndrome.

Description of drawings

With the quantitative accuracy of single standard product and separate standards product relatively, with each standard substance all according to 1.0 * 10 ⁷, 1.0 * 10 ⁶, 1.0 * 10 ⁵, 1.0 * 10 ⁴The copies/ml gradient dilution, carry out quantitative amplification, the amplification curve that the standard substance of each concentration are corresponding is from left to right corresponding one by one successively on amplification figure, and the quantitative accuracy of single standard product and separate standards product has consistence, Fig. 1-8, various sample positive tests:

Fig. 1, HRVA-NVG I-NVG II-HAstV single standard product amplification curve FAM passage;

Fig. 2, HRVA separate standards product amplification curve FAM passage;

Fig. 3, HRVA-NVG I-NVG II-HAstV single standard product amplification curve HEX passage;

Fig. 4, NVG I separate standards product amplification curve HEX passage;

Fig. 5, HRVA-NVG I-NVG II-HAstV single standard product amplification curve ROX passage;

Fig. 6, NVG II separate standards product amplification curve ROX passage;

Fig. 7, HRVA-NVG I-NVG II-HAstV single standard product amplification curve CY5 passage;

Fig. 8, the independent accurate product amplification curve CY5 passage of HAstV;

Fig. 9, A group rotavirus (HRVA), norovirus I group (NVG I), norovirus II group (NVG II) and the positive experimental result of people's Astrovirus (HAstV);

Figure 10, the internal packing of test kit of the present invention consists of and specification: A is 1 of PCR MIX, and specification is 960ul; B is 1, DEPC water, and specification is 2000ul; C is that reversed transcriptive enzyme is 1, and specification is 50ul; D is that the Taq enzyme is 1, and specification is 50ul; E is HRVA-NVG I-NVG II-HAstV single standard product (1.0 * 10 ⁷Copies/ml) 1, specification is 50ul, the user can prepare 1.0 * 10 with 1 * TE buffer or negative quality control product ⁶, 1.0 * 10 ⁵, 1.0 * 10 ⁴The standard substance of copies/ml; 1 of the negative quality control product of F, specification is 250ul.Test kit is furnished with separately one bottle of RNA extracting solution in addition, and specification is 10ml.The specification of test kit is the 48T/ box, and validity period 10 months needs-20 ℃ of preservations, avoids multigelation.

Embodiment

Embodiment 1: the collection of sample and transporting

The applicable sample type that detects comprises patient suspected's stool sample, can be also the samples such as oropharyngeal swab specimen, bleb liquid, cerebrospinal fluid or viral separation and Culture thing.

The collection of stool sample and Transfer method: wipe away with sterile swab and get ight soil or the diarrhoea thing is inserted sterile chamber, sealing is sent to immediately the laboratory and is detected, and wraps up with dry ice in sample fortune process; Perhaps can be stored in a short time-20 ℃, prolonged preservation can be put-70 ℃, can not surpass 6 months to prevent the degraded of viral RNA in principle.

The collection of oropharyngeal swab specimen and Transfer method: swab pharynx rear wall and tonsilla position, both sides secretory product under the throat swab aseptic condition and insert in sampling tube, sealing is sent to immediately the laboratory and is detected, and wraps up with dry ice in sample fortune process; Perhaps can be stored in a short time-20 ℃, prolonged preservation can be put-70 ℃, can not surpass 6 months to prevent the degraded of viral RNA in principle.

The collection of bleb liquid and Transfer method: needle bleb under aseptic condition, cotton swab dips bleb liquid, and sealing is sent to immediately the laboratory and is detected, and wraps up with dry ice in sample fortune process; Perhaps can be stored in a short time-20 ℃, prolonged preservation can be put-70 ℃, can not surpass 6 months to prevent the degraded of viral RNA in principle.Same patient can gather a plurality of blebs simultaneously as a sample.

The collection of cerebrospinal fluid and Transfer method: in the acute phase after neurological symptom occurring in 3 days, collection capacity 1-2ml cerebrospinal fluid.The censorship immediately in aseptic cryopreservation tube of packing into after collection, or-20 ℃ of following cryogenic freezing preservations need the sample of prolonged preservation to be stored in-70 ℃ of refrigerators.

The directly censorship of virus separation and Culture thing, or-20 ℃ of following cryogenic freezing preservations, the sample of prolonged preservation need be stored in-70 ℃ of refrigerators.

Embodiment 2: the extraction of viral RNA in sample

The sample liquid of getting the 100 above-mentioned collections of μ l adds the RNA extracting solution in the 300ul test kit fully to shake, and room temperature is placed 5min; Add the 100ul chloroform, firmly shake 15s, the standing 5min of room temperature, 4 ℃ 13, the centrifugal 10min of 000rpm; Carefully with the upper water phase transition in clean centrifuge tube, add the equal-volume Virahol, abundant mixing, the centrifugal 10min of 13,000rpm; Abandon supernatant, add the DEPC ethanol of 500 μ l 75%, abundant mixing, the centrifugal 10min of 13,000rpm carefully sucks most of ethanol; With extraction tube uncovered in air at room temperature dry 5min treat that ethanol volatilization is clean, with 20 μ l DEPC H2O dissolution precipitations.Get the negative quality control product of 50 μ l and add 300 μ l Trizol RNA extracting solutions fully to shake, room temperature is placed 10min, processes equally by aforesaid operations afterwards.

The preparation of embodiment 3:HRVA-NVG I-NVG II-HAstV single standard product

3.1 the RT-PCR experiment, reverse transcription synthesizes cDNA

Take the RNA of said extracted as template, according to PrimeScript 1st Strand cDNA Synthesis Kit(TAKARA) specification sheets preparation reverse transcription reaction system, be totally 20ul, synthetic cDNA the first chain:

Random primer	1.0ul
		dNTP（10mM）	1.0ul
RNA	5.0ul
		Rnase free dH ₂O	3.0ul
Total	10.0ul

Above-mentioned reaction solution, 65 ℃ of 5min, chilling on ice, then,

Above-mentioned reaction solution	10.0ul
		5×PrimerScript Buffer	4.0ul
RNase Ihibitor（40U/ul）	0.5ul
		PrimerScipt Rtase(200U/ul)	1.0ul
Rnase free dH ₂O	4.5ul
		Total	20.0ul

30 ℃, 10min; 42 ℃, 60min; 70 ℃, 15min, synthetic cDNA the first chain obtains respectively HRVA, NVG I, NVG II and HAstV cDNA masterplate, and collects standby.

3.2 the target fragment of conventional pcr amplification HRVA, NVG I, NVG II and HAstV virus

3.2.1 the amplification of A group rotavirus (HRVA) target fragment (amplification for the first time):

10×PCR buffer	5.0ul
		HRVA-F1、HRVA-R1（50uM）	Each0.2ul
dNTP（10mM）	1.0ul
		Taq（ABI）（5U/ul）	1.0ul
HRVA cDNA	5.0ul
		DDH ₂O	37.8ul
Total	50.0ul

According to 93 ℃, 3min; 93 ℃, 30s, 57 ℃, 45s, 72 ℃, 45s, 35cycles; 72 ℃, 10min; 4 ℃ of preservations.Amplifying target genes.Reaction is got the 5ul amplified production and is carried out 2% agarose gel electrophoresis analysis, and use the gel imaging system Taking Pictures recording after finishing.All the other PCR products add the 3M sodium-acetate of 1/10 volume, the dehydrated alcohol of two volumes, and-20 ℃ are spent the night.12,000 rev/mins centrifugal, removes supernatant, 75% ethanol is washed one time, adds 30 μ l water dissolution, the PCR product after concentrated is carried out 1% agarose gel electrophoresis, and with the QIAquick Gel Extraction Kit test kit purifying of tapping rubber, step is undertaken by process specifications.

The part PCR product of purifying is sent to the order-checking of Invitrogen company, and all the other products-20 ℃ save backup.

3.2.2 the amplification of norovirus I group (NVG I), norovirus II group (NVG II) and people's Astrovirus (HAstV) target fragment is the same.

3.3 the design of HRVA-NVG I-NVG II-HAstV single standard product Auele Specific Primer

according to prioritization scheme of the present invention, the present invention is the quadruple four look fluorescence quantifying PCR methods of single standard product, that is to say for above-mentioned A group rotavirus (HRVA), norovirus I group (NVG I), the standard substance of norovirus II group (NVG II) and four kinds of viruses of people's Astrovirus (HAstV) are single, so at first, each target sequence is analyzed and is introduced the nonsense sequence that is no less than the 6-8 base between each sequence, four sections different standard substance sequences increase, each fragment is between 100-120bp, the joint has the 6-8 of being no less than sequence to repeat, increase for the second time take each viral target fragment as masterplate.Mix as template at the external amplified fragments that all are corresponding and be the sliceable single Long fragment gene that obtains through pcr amplification for the third time, as the nucleotide sequence of single standard product, be cloned into the pMD18-T carrier, be prepared into standard substance.Primer after adjustment is as follows, and lower case is modified base:

For the A group rotavirus:

Upstream primer HRVA-F2:5 '-atgactctgcTGACCCTCTATGA-3 ' (SEQ ID NO.17);

Downstream primer HRVA-R2:5 '-cattgcagtcaCACATAACGC-3 ' (SEQ ID NO.18);

The amplified fragments sequence is:

atgactctgcTGACCCTCTATGAGCACAATAGTTAAAAGCTAACACTGTCAAAAACCTAAATGGCTATAGGGGCGTTATGTGtgactgcaatg（SEQ ID NO.19）；

For norovirus I group:

Upstream primer NVG I-F2:5 '-tgactgcaatgTGGATGCGGTTC-3 ' (SEQ ID NO.20);

Downstream primer NVG I-R2:5 '-gatctcatgcCCTTAGACGCCAT-3 ' (SEQ ID NO.21);

The amplified fragments sequence is:

tgactgcaatgTGGATGCGCTTCCATGATCTCGGATTGTGGACAGGAGATCGCGATCTCCTGCCCGAATTCGTAAATGATGATGCCGTCTAAGGgcatgagatc（SEQ ID NO.22）；

For norovirus II group:

Upstream primer NVG II-F2:5 '-gcatgagatcATTCTCGGA-3 ' (SEQ ID NO.23);

Downstream primer NVG II-R2:5 '-tgcttacaggaTCGACGCCATC-3 ' (SEQ ID NO.24);

The amplified fragments sequence is:

gcatgagatcATTCTCGGACCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGTTTTGTGAATGAAGATGGCGTCGAtcctgtaagca（SEQ ID NO.25）；

For people's Astrovirus:

Upstream primer HAstV-F2:5 '-tcctgtaagcaCATCTGGAAG-3 ' (SEQ ID NO.26);

Downstream primer HAstV-R2:5 '-gatgcgcgctcacgTAAAATAATCTAA-3 ' (SEQ ID NO.27);

The amplified fragments sequence is:

tcctgtaagcaCATCTGGAAGCCGCGGCCACGCCGAGTAGGAACGAGGGTACAGCTTCCTTCTTTTCTGTCTCTGTTTAGATTATTTTAcgtgagcgcgcatc（SEQ ID NO.28）。

3. 4 amplifications (amplification for the second time) of modifying rear HRVA, NVG I, NVG II and HAstV target fragment:

3.4.1 the amplification of A group rotavirus (HRVA) target fragment after modifying (amplification for the second time)

Be primer for the upstream primer HRVA-F2:5 ' of A group rotavirus-atgactctgcTGACCCTCTATGA-3 ', downstream primer HRVA-R2:5 '-cattgcagtcaCACATAACGC-3 ', take the product of the amplification for the first time of A group rotavirus (HRVA) target fragment of 3.2.1 as masterplate, experimental technique with reference to 3.2.1 increases, and with product purification, part PCR product is sent to the order-checking of Invitrogen company, and all the other products-20 ℃ save backup.

So, the template fragment sequence of HRVA-NVG I-NVG II-HAstV single standard product is:

atgactctgcTGACCCTCTATGAGCACAATAGTTAAAAGCTAACACTGTCAAAAACCTAAATGGCTATAGGGGCGTTATGTGtgactgcaatgTGGATGCGCTTCCATGATCTCGGATTGTGGACAGGAGATCGCGATCTCCTGCCCGAATTCGTAAATGATGATGCCGTCTAAGGgcatgagatcATTCTCGGACCTGAGCACGTGGGAGGGCGATCGCAATCTGGCTCCCAGTTTTGTGAATGAAGATGGCGTCGAtcctgtaagcatcctgtaagcaCATCTGGAAGCCGCGGCCACGCCGAGTAGGAACGAGGGTACAGCTTCCTTCTTTTCTGTCTCTGTTTAGATTATTTTAcgtgagcgcgcatc（SEQ ID NO.29）。

3.4.2 the amplification of norovirus I group (NVG I), norovirus II group (NVG II) and people's Astrovirus (HAstV) target fragment after modifying (amplification for the second time) is the same.

3.5 the preparation of HRVA-NVG I-NVG II-HAstV single standard product masterplate

take for the upstream primer HRVA-F2:5 ' of A group rotavirus-atgactctgcTGACCCTCTATGA-3 ' as upstream primer, take for the downstream primer HAstV-R2:5 ' of people's Astrovirus-gatgcgcgctcacgTAAAATAATCTAA-3 ' as downstream primer, with A group rotavirus (HRVA) after 3.4 modification, norovirus I group (NVG I), the amplification of norovirus II group (NVG II) and four kinds of target fragments of people's Astrovirus (HAstV) (amplification for the second time) product is masterplate, experimental technique with reference to 3.2.1 increases, and with product purification, part PCR product is sent to the order-checking of Invitrogen company, all the other products-20 ℃ save backup.

It is the masterplate for HRVA-NVG I-NVG II-HAstV single standard product.

3.6 the preparation of HRVA-NVG I-NVG II-HAstV single standard product

3.6.1 the PCR product of purifying and the ligation of T carrier

The masterplate of single standard product	50.0ng
		pMD 18-T Vector	50.0ng
10×T4 DNA Ligase Buffer	2.0ul
		T4 DNA Ligase	1.0ul
DDH ₂O	Supply
		Total	20.0ul

Reaction conditions: 16 ℃ 3 hours, carry out ligation.

3.6.2 the preparation of competent cell

Picking list colony inoculation is in the 5mlLB nutrient solution from the DH5 а culture dish of 37 ℃ of overnight incubation, 37 ℃ of shaking culture 12 hours, get 1ml bacterium liquid and be seeded in 100ml LB nutrient solution, it is that 0.3-0.4(approximately needs 2-3 hour that 37 ℃ of joltings are cultured to bacterium liquid OD600), divide to be filled in two large centrifuge tubes of 50ml, ice bath 30 minutes, 3,000 rev/mins, 4 ℃ centrifugal 10 minutes, abandon supernatant, with the 0.1mol CaCl of 10ml ice precooling ₂Resuspended thalline, ice bath 30 minutes, 4,000 rev/mins, 4 ℃ centrifugal 10 minutes, abandon supernatant, with the 0.1mol CaCl of 2ml ice precooling ₂Resuspended thalline, packing 100 μ l/ pipes were put 4 ℃ after 14 hours, and-80 ℃ are frozen standby.

3.6.3 the conversion of HRVA-NVG I-NVG II-HAstV-pMD 18-T recombinant plasmid

To connect product 10 μ l and join mixing in 100 μ l competent cells, ice bath 30 minutes, 42 ℃ of heat-shockeds 90 seconds, ice bath.Add LB substratum 200 μ l, 37 ℃ of gentle vibrations 45 minutes, to contain simultaneously on penbritin (100 μ g/ml) agar culture dish and evenly coat 4 μ l IPTG and 40 μ l X-gal, above mixture will evenly be coated on culture dish, and be inverted for 37 ℃ and cultivated 12-16 hour.

3.6.4 extraction and the evaluation of HRVA-NVG I-NVG II-HAstV-pMD 18-T recombinant plasmid

Get the positive bacterium colony extracting plasmid of PCR reaction, operation is undertaken by QIAGEN Plasmid Midi Kit short run plasmid DNA purification system test kit specification sheets.

Take above-mentioned plasmid as masterplate take for the upstream primer HRVA-F2:5 ' of A group rotavirus-atgactctgcTGACCCTCTATGA-3 ' as upstream primer, to identify as downstream primer carries out PCR for the downstream primer HAstV-R2:5 ' of people's Astrovirus-gatgcgcgctcacgTAAAATAATCTAA-3 ', and with product purification, part PCR product is sent to the order-checking of Invitrogen company, all the other products-20 ℃ preservation.

It is the recombinant plasmid for HRVA-NVG I-NVG II-HAstV single standard product.

Finally, the recombinant plasmid that the clone is successful is got 100 times of 5 μ l dilutions, surveys its OD value and calculates its concentration, then according to molecular size range, be converted into molecule number, becomes 1 * 10 with the sterilization pure water by 10 times of gradient dilutions ⁷Copy/ml further demarcates with the other standards product of concentration known, and definite value is (1 * 10 ⁷Copies/ ml), dilution is 1 * 10 successively ⁷, 1.0 * 10 ⁶, 1.0 * 10 ⁵, 1.0 * 10 ⁴Copies/ml is as final plasmid standards for quantitation.

To be HRVA-NVG I-NVG II-HAstV single standard product.

3.7 the preparation of HRVA standard substance, standard substance NVG I standard substance, NVG II standard substance and four kinds of separate standards product of HAstV standard substance

The PCR product of HRVA, NVG I, NVG II and the HAstV target fragment that increases respectively take 3.2.1 is as masterplate, with reference to and with product purification, part PCR product is sent to the order-checking of Invitrogen company, all the other products-20 ℃ preservation.To be HRVA, NVG I, NVG II and HAstV separate standards product template.

Above-mentioned separate standards product template is prepared HRVA, NVG I, NVG II and four kinds of standard substance of HAstV according to 3.6 methods that are.

To be HRVA, NVG I, NVG II and four kinds of separate standards product of HAstV.

Embodiment 4: the instrument that this detection method and test kit are applicable

single stage method A group rotavirus (HRVA) based on the single standard product of the present invention, norovirus I group (NVG I), the applicable instrument of norovirus II group (NVG II) and people's Astrovirus (HAstV) RNA quadruple four look fluorescent quantitative PCR detection methods and test kit mainly comprises: the Stratagene Mx3000P/Mx3005P of company, ABIGeneAmp PCR System7500, ABI PRISM 7300, ABI GeneAmp PCR System7700, the LightCycler quantitative real time PCR Instrument, 4 passage quantitative real time PCR Instruments of MJ Opticon series and other authentications that qualify.

Embodiment 5: quadruple four look fluorescence PCR methods to HRVA, NVG I, NVG II and, HAstV RNA detects

The preparation of PCR system and application of sample:

Take out PCR MIX, Taq enzyme system from test kit, reversed transcriptive enzyme system, room temperature melt and the mixing that vibrates after, the centrifugal 10s of 10,000rpm.Calculate the amount of needed PCR MIX, according to each detection:

PCR MIX	20μl
		Taq enzyme system	1μl
Reversed transcriptive enzyme system	1μl

The preparation system, abundant mixing, then packing 22 μ l PCR MIX in each PCR reaction tubes of instantaneous centrifugal backward setting add the rear sample RNA+HRVA-NVG I-NVG II-HAstV single standard product (1 * 10 of processing ⁷, 1.0 * 10 ⁶, 1.0 * 10 ⁵, 1.0 * 10 ⁴Copies/ml)+negative quality control product 3 μ l, instantaneous centrifugal after, put into the reactive tank of quantitative PCR instrument, by correspondence, positive criteria product, negative quality control product and sample to be measured are set sequentially, each reactive tank is selected FAM, HEX, ROX and CY5 fluorescence channel simultaneously, respectively the detection of corresponding A group rotavirus (HRVA), norovirus I group (NVG I), norovirus II group (NVG II) and people's Astrovirus (HAstV) RNA.

Amplification condition arranges:

The Mx3000P of Stratagene company and Mx3005P etc., ABI PRISM7300/7500, ABI PRISM 7700, ABI PRISM5700, ABIGeneAmp 7000, MJ Opticon etc. use quantitative real time PCR Instrument, cycling condition is 42 ℃ → 20 minutes, then 93 ℃ → 2 minutes, then 93 ℃ 30 seconds → 55 ℃ 45 seconds (gathering fluorescent signal in this step), 40 circulations; The fluorescence such as LightCycler are decided the quantitative PCR instrument, and cycling condition is 42 ℃ → 20 minutes, and 93 ℃ → 2 minutes, then 93 ℃ 5 seconds → 55 ℃ gathered fluorescent signal, totally 40 circulations in this step in 45 seconds.

Embodiment 6: interpretation of result and judgement:

After reaction finishes, at first select a kind of fluorescence (FAM, HEX, ROX and CY5 be corresponding A group rotavirus, norovirus I group, norovirus II group and people's Astrovirus respectively), use the manual setting threshold value to make negative quality control product Ct value more than 40, the Ct value is certain virus-positive less than 35 circulations; The Ct value for suspicious, need to be carried out revision test between 35-40 circulation.After revision test, if the Ct value still between 35-40 circulation, is certain virus-positive, do not have fluorescent value to increase negative.

In addition, by HRVA-NVG I-NVG II-HAstV single standard product (1 * 10 ⁷, 1.0 * 10 ⁶, 1.0 * 10 ⁵, 1.0 * 10 ⁴Can realize copies/ml) carried out quantitatively the load of a certain virus in sample.

The prerequisite of judged result is: negative quality control product should be total negative; The Ct value of positive criteria product all should be less than 30, and are standard S type amplification curve.The typical curve equidistant parallel of HRVA-NVG I-NVG II-HAstV single standard product.

Embodiment 7: the clinical detection of the inventive method is used

Use the quadruple four look PCR kit for fluorescence quantitative of the A group rotavirus based on the single standard product of the present invention, norovirus I group, norovirus II group and people's Astrovirus that a large amount of samples are detected, and finally through the order-checking experimental verification.Result shows: test kit accuracy and the specificity of the inventive method reach 100%, and repeatability and stability are better, and the preservation condition of this test kit and validity period are :-20 ℃, and 10 months.

Test kit of the present invention carries out four kinds of Detectings for having realized a reaction to a sample, and method is simple and efficient, is a kind of high-throughout detection method.

The present invention is based on the single standard product as positive control and the quantitative criterion of experiment, the above-mentioned viral RNA that infects is carried out rapid detection and quantitative, improved the efficient that experiment detects, reduced the chance of polluting.Can monitor for laboratory high throughput testing and the epidemic situation of the rotavirus, norovirus and the Astrovirus that cause the diarrhoea syndrome quickly and easily.

SEQUENCE LISTING

＜110〉the prosperous bio tech ltd of Guangzhou dimension uncle

＜120〉a kind of quadruple four look fluorescent quantitative PCR detection method and test kits based on the single standard product

<130>

<160> 29

<170> PatentIn version 3.5

<210> 1

<211> 24

<212> DNA

＜213〉artificial sequence

<400> 1

tgaccctcta tgagcacaat agtt 24

<210> 2

<211> 20

<212> DNA

＜213〉artificial sequence

<400> 2

cacataacgc ccctatagcc 20

<210> 3

<211> 25

<212> DNA

＜213〉artificial sequence

<400> 3

aaagctaaca ctgtcaaaaa cctaa 25

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<211> 17

<212> DNA

＜213〉artificial sequence

<400> 4

tggatgcgbt tccatga 17

<210> 5

<211> 23

<212> DNA

＜213〉artificial sequence

<400> 5

ccttagacgc catcatcatt tac 23

<210> 6

<211> 25

<212> DNA

＜213〉artificial sequence

<400> 6

caggagatcg caatctcctg cccga 25

<210> 7

<211> 20

<212> DNA

＜213〉artificial sequence

<400> 7

attctcdgay ctsagcacrt 20

<210> 8

<211> 20

<212> DNA

＜213〉artificial sequence

<400> 8

tcgacgccat cttcattcac 20

<210> 9

<211> 23

<212> DNA

＜213〉artificial sequence

<400> 9

agggcgatcg caatctggct ccc 23

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<212> DNA

＜213〉artificial sequence

<400> 10

catctggaag ccgcgg 16

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<211> 28

<212> DNA

＜213〉artificial sequence

<400> 11

taaaataatc taaacagaga cagaaaag 28

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<211> 28

<212> DNA

＜213〉artificial sequence

<400> 12

cycacgccga gtaggaacga gggtacag 28

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<212> DNA

＜213〉HRVA target sequence

<400> 13

tgaccctcta tgagcacaat agttaaaagc taacactgtc aaaaacctaa atggctatag 60

gggcgttatg tg 72

<210> 14

<211> 83

<212> DNA

＜213〉NVG I target sequence

<400> 14

tggatgcgct tccatgatct cggattgtgg acaggagatc gcgatctcct gcccgaattc 60

gtaaatgatg atgccgtcta agg 83

<210> 15

<211> 72

<212> DNA

＜213〉NVG II target sequence

<400> 15

attctcggac ctgagcacgt gggagggcga tcgcaatctg gctcccagtt ttgtgaatga 60

agatggcgtc ga 72

<210> 16

<211> 78

<212> DNA

＜213〉HAstV target sequence

<400> 16

catctggaag ccgcggccac gccgagtagg aacgagggta cagcttcctt cttttctgtc 60

tctgtttaga ttatttta 78

<210> 17

<211> 23

<212> DNA

＜213〉artificial sequence

<400> 17

atgactctgc tgaccctcta tga 23

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<211> 21

<212> DNA

＜213〉artificial sequence

<400> 18

cattgcagtc acacataacg c 21

<210> 19

<211> 93

<212> DNA

＜213〉HRVA modification sequence

<400> 19

atgactctgc tgaccctcta tgagcacaat agttaaaagc taacactgtc aaaaacctaa 60

atggctatag gggcgttatg tgtgactgca atg 93

<210> 20

<211> 23

<212> DNA

＜213〉artificial sequence

<400> 20

tgactgcaat gtggatgcgg ttc 23

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<211> 23

<212> DNA

＜213〉artificial sequence

<400> 21

gatctcatgc ccttagacgc cat 23

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<211> 104

<212> DNA

＜213〉NVG I modification sequence

<400> 22

tgactgcaat gtggatgcgc ttccatgatc tcggattgtg gacaggagat cgcgatctcc 60

tgcccgaatt cgtaaatgat gatgccgtct aagggcatga gatc 104

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<212> DNA

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gcatgagatc attctcgga 19

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<400> 24

tgcttacagg atcgacgcca tc 22

<210> 25

<211> 93

<212> DNA

＜213〉NVG II modification sequence

<400> 25

gcatgagatc attctcggac ctgagcacgt gggagggcga tcgcaatctg gctcccagtt 60

ttgtgaatga agatggcgtc gatcctgtaa gca 93

<210> 26

<211> 21

<212> DNA

＜213〉artificial sequence

<400> 26

tcctgtaagc acatctggaa g 21

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<211> 27

<212> DNA

＜213〉artificial sequence

<400> 27

gatgcgcgct cacgtaaaat aatctaa 27

<210> 28

<211> 103

<212> DNA

＜213〉HAstV modification sequence

<400> 28

tcctgtaagc acatctggaa gccgcggcca cgccgagtag gaacgagggt acagcttcct 60

tcttttctgt ctctgtttag attattttac gtgagcgcgc atc 103

<210> 29

<211> 372

<212> DNA

＜213〉the template fragment sequence of HRVA-NVG I-NVG II-HAstV single standard product

<400> 29

atgactctgc tgaccctcta tgagcacaat agttaaaagc taacactgtc aaaaacctaa 60

atggctatag gggcgttatg tgtgactgca atgtggatgc gcttccatga tctcggattg 120

tggacaggag atcgcgatct cctgcccgaa ttcgtaaatg atgatgccgt ctaagggcat 180

gagatcattc tcggacctga gcacgtggga gggcgatcgc aatctggctc ccagttttgt 240

gaatgaagat ggcgtcgatc ctgtaagcat cctgtaagca catctggaag ccgcggccac 300

gccgagtagg aacgagggta cagcttcctt cttttctgtc tctgtttaga ttattttacg 360

tgagcgcgca tc 372

Claims

1. the fluorescent quantitative PCR technique of quadruple four looks of the present invention based on the single standard product, it is characterized in that adopting the single standard product to detect after all kinds of processing A group rotavirus (HRVA), norovirus I group (NVG I), norovirus II group (NVG II) and people's Astrovirus (HAstV) RNA in sample as the positive control of experiment and quantitative criterion by the single stage method pcr amplification, the laboratory fast high-flux that is used for causing common A rotavirus, norovirus I group, norovirus II group and people's Astrovirus of diarrhoea syndrome detects and epidemic situation is monitored.

2. a kind of detection method according to claim 1, detection method experimental procedure of the present invention comprises: 1) infect or doubtful sample collection and transporting; 2) sample preprocessing and extraction RNA; 3) single stage method quadruple four look quantitative fluorescent PCR samples detect; 4) carry out accurate quantification according to the virus in the amplification curve sample of single standard product.

3. detection method according to claim 1 and test kit for detection of A group rotavirus (HRVA), norovirus I group (NVG I), norovirus II group (NVG II) and the specific primer of people's Astrovirus (HAstV) and fluorescent probe are: A group rotavirus upstream primer HRVA-F1(SEQ ID NO.1), downstream primer HRVA-R1(SEQ ID NO.2) and fluorescent probe HRVA-P(SEQ ID NO.3); Norovirus I group upstream primer NVG I-F1(SEQ ID NO.4), downstream primer NVG I-R1(SEQ ID NO.5) and fluorescent probe NVG I-P(SEQ ID NO.6); Norovirus II group upstream primer NVG II-F1(SEQ ID NO.7), downstream primer NVG II-R1(SEQ ID NO.8) and fluorescent probe NVG II-P(SEQ ID NO.9); People's Astrovirus upstream primer HAstV-F1(SEQ ID NO.10), downstream primer HAstV-R1(SEQ ID NO.11) and fluorescent probe HAstV-P(SEQ ID NO.12).

4. Auele Specific Primer according to claim 3 and probe, the target region that the quadruple four look quantitative fluorescent PCR specific amplifications of four kinds of viral RNAs detect is respectively: A group rotavirus (SEQ ID NO.13), norovirus I group (SEQ ID NO.14), norovirus II group (SEQ ID NO.15) and people's Astrovirus (SEQ ID NO.16).

5. a kind of test kit according to claim 1, for A group rotavirus (HRVA), norovirus I group (NVG I), the nonsense sequence is analyzed and introduced to the target sequence of norovirus II group (NVG II) and four kinds of viruses of people's Astrovirus (HAstV) between each sequence, design 4 sections different standard substance sequences, each fragment is between 100-120bp, the joint has the 6-8 of being no less than sequence to repeat, increase for the second time take viral target fragment as masterplate, mixing as template at the external amplified fragments that all are corresponding is the sliceable single Long fragment gene that obtains through pcr amplification for the third time, nucleotide sequence as the single standard product, be cloned into the pMD18-T carrier, be prepared into standard substance.

6. the quadruple four look fluorescent quantificationally PCR detecting kits of a kind of single standard product according to claim 1, preparing the primer of using that this kind be prepared into standard substance is A group rotavirus upstream primer HRVA-F2(SEQ ID NO.17) and downstream primer HRVA-R2(SEQ ID NO.18), amplified fragments is (SEQ ID NO.19); Norovirus I group upstream primer NVG I-F2(SEQ ID NO.20) and downstream primer NVG I-R2(SEQ ID NO.21), the amplified fragments sequence is (SEQ ID NO.22); Norovirus II group upstream primer NVG II-F2(SEQ ID NO.23), downstream primer NVG II-R2(SEQ ID NO.24), the amplified fragments sequence is (SEQ ID NO.25); For people's Astrovirus upstream primer HAstV-F2(SEQ ID NO.26) and downstream primer HAstV-R2(SEQ ID NO.27), the amplified fragments sequence is (SEQ ID NO.28), and finally the template fragment sequence through the HRVA-NVG I that increases for the third time-NVG II-HAstV single standard product is (SEQ ID NO.29).

7. wherein said test kit according to claim 1, the preparation in the following manner of reaction system (PCR MIX): every part of reaction solution (PCR MIX) volume is 20.0ul, then adding reversed transcriptive enzyme is that 1.0ul, Taq enzyme are 1.0ul, RNA/ standard substance/negative control 3.0ul, need not to increase through carrying out separately the direct single stage method of reverse transcription reflection.

8. quadruple PCR kit for fluorescence quantitative according to claim 1, each reactive tank is selected FAM, HEX, ROX and CY5 fluorescence channel simultaneously, respectively the detection of corresponding A group rotavirus (HRVA), norovirus I group (NVG I), norovirus II group (NVG II) and people's Astrovirus (HAstV) RNA.

9. quadruple PCR kit for fluorescence quantitative according to claim 1, Mx3000P and Mx3005P, ABI PRISM7300/7500, ABI PRISM 7700, ABI PRISM5700, ABI GeneAmp 7000, the MJ Opticon etc. of use Stratagene company use quantitative real time PCR Instruments, amplification condition is 42 ℃ → 20 minutes, then 93 ℃ → 2 minutes, then 93 ℃ 30 seconds → 55 ℃ 45 seconds (gathering fluorescent signal in this step), 40 circulations.