CN102605103B

CN102605103B - Primer, probe and method for detecting Norwalk virus nucleotide

Info

Publication number: CN102605103B
Application number: CN2012100681121A
Authority: CN
Inventors: 邱杨; 黄伟; 卢小雨; 刘建丽; 余以刚; 陈进会; 赵丽
Original assignee: Inspection & Quarantine Comprehensive Technology Center Dongguan Entry-Exit Inspection And Quarantine; Animal and Plant Inspection and Quarantine Technology Center of Shenzhen Entry Exit Inspection and Quarantine Bureau
Current assignee: Inspection & Quarantine Comprehensive Technology Center Dongguan Entry-Exit Inspection And Quarantine; Animal and Plant Inspection and Quarantine Technology Center of Shenzhen Entry Exit Inspection and Quarantine Bureau
Priority date: 2012-03-15
Filing date: 2012-03-15
Publication date: 2013-12-04
Anticipated expiration: 2032-03-15
Also published as: CN102605103A

Abstract

The invention discloses a primer, a probe and a method for detecting Norwalk virus nucleotide, belonging to the technical field of virus detection. The nucleotide sequences of the primer and the probe are respectively shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 in a sequence table.In the method for detecting Norwalk virus nucleotide disclosed by the invention, a sample RNA (ribonucleic acid) is taken as a template, the primer and the probe are used for carrying out real-time fluorescent RT-PCR (reverse transcriptase-polymerase chain reaction) amplification, and data is acquired after each circulation is finished; and after reaction is finished, and a Norwalk virus specificity fluorescence amplification curve appears, thereby determining that the Norwalk virus nucleotide is contained in the sample RNA.

Description

A kind of primer, probe and method that detects Norwalk virus Nucleotide

Technical field

The invention belongs to the virus detection techniques field, be specifically related to a kind of primer, probe and method that detects Norwalk virus Nucleotide.

Background technology

Norwalk virus (Norwalk virus, NV) be the roundlet shape structure virus (SRSV) of a kind of diameter 27nm, norwalk group viruses (Norwalk-like Viruses is otherwise known as, NLVs), belonging to the Caliciviridae Norwalk virus and belong to the member, is a kind of distribution enteron aisle diarrhea virus very widely.Gastro-enteritis outburst stream has together occurred in nineteen sixty-eight in U.S. Norwalk town, this virus is to be found first in these patients' ight soil, hence obtains one's name.NLV is the main pathogen of the acellular bacterium property gastro-enteritis of eruption and prevalence in community, school, office, military camp and family, infect object and be mainly the adult and school age the ear child, generation is all arranged the whole year.The circulation way of Norwalk virus is to propagate by fecal oral route, is mainly that water source, food and people by polluting-people's close contact is propagated; Clinical manifestation comprises nauseating, vomit, suffer from abdominal pain, suffer from diarrhoea (watery stool), can cut and go down with headache, low-heat, weak and food, the general 2-3d of the course of disease, other viral diarrheas that cause with rotavirus etc. are compared, and the diarrhoea that NLV causes is many with symptoms of emesis.Therefore, high specificity is badly in need of in Disease Prevention and Control Institutions and hospital, and susceptibility is high, easy to operate, can be used for the method for the Rapid&Early diagnosis of the epidemic outbreaks such as gastro-enteritis and clinical case.

Norwalk virus (Norwalk virus, NV) genome is positive single stranded RNA, total length 7.65Kb, 3 opening code-reading frame (open reading frarnes encode, ORFs), the Nonstructural Protein precursor that Nonstructural Protein, coat protein and the not clear albumen of a kind of function: ORFs1 coding have RNA polymerase activity of encoding respectively is Nonstructural Protein; The ORFs2 capsid protein of encoding; ORFs3 a kind of strong basicity albumen of encoding, with the sequence of any protein in gene pool, all without similarity, its function it be unclear that.The Norwalk virus member is numerous and jumbled, its more than 100 strain isolateds is carried out to gene sequencing at present.According to the homology of RNA polymerase zone capsid protein district's Nucleotide and aminoacid sequence relatively, Norwalk virus is divided into to 2 genomes: genome I, representative strains NV, comprise SV etc., Desert Shield virus (DSV) etc.; Gene II group, representative strains SMV, comprise HV, Mexio virus (MX) etc.

The conventional sense method of Norwalk virus is mainly electron microscopy (EM), radioimmunoassay test (RIA) and euzymelinked immunosorbent assay (ELISA) (ELISA) at present.Norwalk virus was found in the rear a very long time, Electronic Speculum is the Main Means detected always, there is direct, reliable advantage, but lack significant morphological feature due to Norwalk virus under Electronic Speculum, and susceptibility is low, expensive, the technical qualification requirement is high, is not suitable for inspection and quarantine real work demand; Detect antibody between acute phase and decubation by the RIA method and raise more than 4 times, the sick investigation of pop is more meaningful, but RIA method experimental period needs the 6d time, also needs labelled with radioisotope simultaneously, and operator and environment are caused to detrimentally affect; Euzymelinked immunosorbent assay (ELISA) (ELISA) high specificity, highly sensitive, diagnosis is rapid, and less expensive, but is the detection method of widespread use at present, but due to the also success of Norwalk virus vitro culture, the virus antigen quantity of available reagent is restricted.Traditional RI-PCR also is used to Norwalk virus, but needs 6 hours detection time, after thermal cycling, will detect analysis to amplified production with electrophoresis, and complex steps also can be crossed in kind and likely pollute in experimental implementation; The advantages such as that real-time fluorescence RT-PCR (real-time fluorescent RT-PCR) technology has is highly sensitive, high specificity, speed are fast, antipollution, high-throughput have replaced traditional RT-PCR technology gradually.

Summary of the invention

The object of the present invention is to provide a kind of primer, probe and method that detects Norwalk virus Nucleotide.

A kind of primer that detects Norwalk virus Nucleotide, its nucleotide sequence is as sequence table SEQ ID No.1, shown in SEQ ID No.2.

A kind of probe that detects Norwalk virus Nucleotide, its nucleotide sequence is as shown in sequence table SEQ ID No.3; 5 ' end of this probe is marked with the report fluorescence dye, and 3 ' end is marked with the cancellation fluorescence dye.

Described report fluorescence dye is FAM, HEX, TET, JOE, VIC, FITC, CY3 or CY5, and described cancellation fluorescence dye is TAMRA, ROX, DABCY1, BHQ1 or BHQ2.

A kind of method that detects Norwalk virus Nucleotide, the method be take sample RNA as template, utilize as sequence table SEQ ID No.1, probe shown in primer shown in SEQ ID No.2 and sequence table SEQ ID No.3 carries out the real-time fluorescence RT-PCR amplification, each loop ends image data, after reaction finishes, if occur, Norwalk virus specificity fluorescent amplification curve is judged in sample RNA contains Norwalk virus Nucleotide.

The reaction system of described real-time fluorescence RT-PCR amplification is as follows: 2.5 μ L 10 * One Step RNA PCR Buffer, the dNTP that final concentration is 0.4mM, the primer that final concentration is 400 μ M, the probe that final concentration is 200 μ M, the Taq enzyme that final concentration is 2.5U, the RNase Inhibitor that final concentration is 20U, the AMV RTase XL that final concentration is 2.5U, 5 μ L sample RNA, 10.5 μ L RNase Free dH ₂O.

The reaction conditions of described real-time fluorescence RT-PCR amplification is as follows: 50 ℃ of reverse transcription 20min; 95 ℃ of sex change 3min; With 95 ℃ of 5s, 45 circulations of 60 ℃ of 40s amplifications.

The present invention is according to the TaqMan technology, added a nucleotide probe that is marked with two fluorescence dye groups on the basis of conventional PCR, for example will report 5 ' end of the probe of fluorochrome label, the cancellation fluorochrome label is at 3 ' end of probe, both form the energy transfer organization, report that the fluorescence that fluorescence dye is launched can be quenched the fluorescence dye absorption, when the two is far away apart from change, restraining effect weakens, and report fluorescence dye signal strengthens.In the amplified reaction process, probe is hybridized with the purpose amplified fragments on template, because the Taq enzyme has the 5 prime excision enzyme activity of 5 ' end to 3 ' end, in the amplification extension stage, probe is cut off, the restraining effect of cancellation fluorescence dye disappears, and report fluorescence dye signal strengthens, in the amplification extension stage, probe is cut off, the restraining effect of cancellation fluorescence dye disappears, and report fluorescence dye signal strengthens, thereby realizes the detection to Norwalk virus.

Beneficial effect of the present invention: the present invention is good according to the primer specificity of Norwalk virus genome sequence design, and that for fluorescent PCR, detects is highly sensitive.Detection method accuracy of the present invention is high, highly sensitive, its can be fast simply judgement sample whether Norwalk virus is arranged, for etiological diagnosis and the differential diagnosis of people's diarrhoea, gastro-enteritis provides scientific basis.

The accompanying drawing explanation

Fig. 1 is the specificity experimental result picture that real-time fluorescence RT-PCR detects Norwalk virus nucleic acid.

Fig. 2 is the detected result figure of the sensitivity experiment of detection method of the present invention.

Embodiment

Below in conjunction with the drawings and specific embodiments, the present invention will be further described.

The design of embodiment 1 primer and probe and synthetic

By respectively all known Norwalk virus genome sequences being compared to analysis, selection is without the section (its sequence is shown in appendix) of secondary structure and high conservative, utilize biosoftware Primer Express3.0 for this section design primer and Taqman probe, and logical this ncbi database has carried out the checking of Blast sequence analysis.Primer sequence is:

Forward primer: 5 '-TCGACGCCATCTTCATTCACA-3 ' (SEQ ID No.1);

Reverse primer: 5 '-CCAATGTTCAGATGGATGAGATTCTC-3 ' (SEQ ID No.2);

The sequence of probe is: 5 '-ATTGCGATCGCCCTCCCACGT-3 ' (SEQ ID No.3);

The VIC fluorophor mark for 5 ' end of this probe, cancellation fluorescence dye BHQ2 mark for 3 ' end.

Embodiment 2 Real-time RT-PCR

(1) extraction of RNA

The extracting of viral sample RNA is extracted with Roche High Pure viral RNA kit test kit, gets 0.5 gram left and right stool sample, adds the suspension that TE 500 μ L make 10-20%, centrifugal 5 minutes of 8000rpm; Get on 200 μ L samples and reset and add 400 μ L in conjunction with liquid, mix, add in the purifying strainer tube 1000rpm, 15s; Discard filtered liquid, change a new collection tube, add 500 μ L inhibitor removal buffer, centrifugal 1 minute of 10000rpm; Discard filtered liquid, change a new collection tube, add 450 μ L washing lotions, centrifugal 1 minute of 10000rpm; Heavily wash once; Finally centrifugal, 13000rpm, 10s, discard remaining washing lotion; Remove collection tube, change the clean Eppendrof of the 1.5ml without a RNA centrifuge tube, add in strainer tube with 50 μ L elutriants, centrifugal 1 minute of 10000rpm, move on to the RNA of extraction in a new centrifuge tube, in-80 ℃ of preservations.

(2) foundation of amplification method

Real-time fluorescence RT-PCR reaction system (table 1):

Table 1 fluorescence quantitative RT-RCR reaction system

Component	25 μ L volumes	Final concentration
			10×One step RNA PCR Buffer	2.5	1×
DNTPs (each 2.5mM)	2	0.4mM
			Forward and reverse primer (each 10 μ M)	Each are 1.5 years old	0.6μM
Probe (10 μ M)	0.5	0.2μM
			The Taq enzyme	0.5	2.5U
RNase Inhi bitor(40U/μL)	0.5	20U
			AMV RTase XL(5U/μL)	0.5	2.5U
RNA	5
			RNase Free dH ₂O	10.5

Annotate: a. is when the Fluorescence PCR volume is different, and each reagent should be adjusted in proportion.

The real-time fluorescence RT-PCR reaction conditions:

After sample hose is put into to ABI company 7500 fluorescent PCR instrument, following condition is set and is reacted: 50 ℃ of reverse transcription 20min; 95 ℃ of sex change 3min; With 95 ℃ of 5s, 60 ℃ of 40s amplification 45 circulations (collection fluorescence).After reaction finishes according to the curve result of determination.

(3) fluorescence RT-PCR

To 10 strains, different human virus is detected the fluorescence RT-PCR reaction system that utilization is set up, detected result as shown in Figure 1,3 strain NV viruses (are numbered NV-SZ01, NV-SZ02, NV-SZ03) corresponding specificity fluorescent amplification curve (referring to A indication in figure) all occurs, be positive.And other 7 strain human virus (the gray nucleus virus Polio I, Polio II, the PolioIII that comprise 3 strain deactivations, 3 strain rotaviruss, 1 strain people Astrovirus.) all do not have fluorescent signal to produce (referring to B indication in figure), be judged to feminine gender.Result shows, designed primer probe is only special to purpose virus (that is, NV virus), with other detected object no cross reaction.

The RNA of Norwalk virus of take is template, detect and can be observed obvious fluorescence intensity change by real-time fluorescence RT-PCR, and other viral fluorescence intensity does not change.

Embodiment 3 sensitivity experiments

The water that the outer transcribe rna of NV virosome is processed with DEPC dilutes respectively, carry out the relative sensitivity detection, in 25 μ L reaction systems, RNA is diluted into respectively 5.0 * 105,5.0 * 104,5.0 * 103,5.0 * 102,5.0 * 101 copy numbers, as shown in Figure 2, result confirms that the lowest detectable limit that Norwalk virus detects all reaches 50 template copies to detected result.

Claims

1. a primer that detects Norwalk virus Nucleotide, is characterized in that, its nucleotide sequence is as sequence table SEQ ID No.1, shown in SEQ ID No.2.

2. a probe that coordinates the described primer of claim 1 to detect Norwalk virus Nucleotide, is characterized in that, its nucleotide sequence is as shown in sequence table SEQ ID No.3; 5 ' end of this probe is marked with the report fluorescence dye, and 3 ' end is marked with the cancellation fluorescence dye.

3. detect according to claim 2 the probe of Norwalk virus Nucleotide, it is characterized in that, described report fluorescence dye is FAM, HEX, TET, JOE, VIC, FITC, CY3 or CY5, and described cancellation fluorescence dye is TAMRA, ROX, DABCY1, BHQ1 or BHQ2.