CN102304514B

CN102304514B - Primer and method for detecting GI type and GII type norovirus by utilizing primer

Info

Publication number: CN102304514B
Application number: CN2011102975515A
Authority: CN
Inventors: 韩建康; 张红河; 吴晓芳; 纪蕾; 徐德顺; 汤仁树
Original assignee: Huzhou Center For Disease Control & Prevention
Current assignee: Huzhou Center For Disease Control & Prevention
Priority date: 2011-01-27
Filing date: 2011-10-08
Publication date: 2012-10-10
Anticipated expiration: 2031-10-08
Also published as: CN102304514A

Abstract

The invention relates to a primer and a method for detecting GI type and GII type norovirus by utilizing the primer, belonging to the technical field of biological detection. The sequence of the primer is as follows: Nov-F: 5-GTGAATGAWGATGGCGTCKA-3, Nov-R: 5-GTHAGWATCCAGGGGTCAAT-3. The invention focuses on the design of the sequence of the primer, the composition of an RT-PCR (reverse transcription-polymerase chain reaction) system, the selection of reaction conditions and the judgment of reaction results can be performed according to the conventional methods in the field. By adopting the primer and the method provided by the invention, a pair of primers can simultaneously detect and differentiate the GI type and the GII type norovirus in a same reaction tube, thereby not only simplifying the operation steps, but also shortening the detection time.

Description

A kind of primer and utilize this primer to detect the method for G I type G II type norovirus

Technical field

The present invention relates to a kind of primer and utilize this primer to detect the method for G I type G II type norovirus, belong to technical field of biological.

Background technology

(Noroviruses NVs) belongs to the Caliciviridae norovirus and belongs to norovirus, is that the whole world causes the important cause of disease that nonbacterial gastroenteritis breaks out and distributes.At present according to virogene RNA polymerase (RNA dependent RNA polymerase; RdRp) and the nucleotide sequence difference of main capsid protein VP1; Norovirus can be divided into 5 heredity group (Genogroup I～V; G I～V), what wherein cause the human infection mainly is G I type and G II type norovirus, and each heredity group can further be divided into several genotype again.Because this viral resistance is strong, cause a disease that dosage is low, each age level crowd is to its general susceptible, often can through routes of transmission such as contaminated food or water source in some public places such as school, kindergarten, hospital etc. cause large-scale outbreak of epidemic.Therefore set up simple, quick, special sensitive detection method, and distinguish norovirus G I type and G II type significant to these sick prevention and control.

Because norovirus can not carry out cell and tissue culture, does not also have suitable animal model, its detection method is very limited.Be used for this viral detection method at present and mainly contain electron microscopy, Immunological Method and molecular biology method.Electron microscopy has directly, reliable advantage, but detection sensitivity is lower, and the stool sample virus titer reaches 10 usually ⁶/ g just can reach the detection requirement.

Summary of the invention

The objective of the invention is provides a kind of primer for solving the problems of the technologies described above.

Above-mentioned technical purpose of the present invention is achieved through following technical scheme.

A kind of primer, its sequence is following:

Nov-F：5-GTGAATGAWGATGGCGTCKA-3 (nt5353-nt5372)

Nov-R：5-GTHAGWATCCAGGGGTCAAT-3 (nt5528-nt5510)。

It described in the bracket the segmental nucleotide position of the corresponding purpose of this primer.

Another object of the present invention provides a kind of method of utilizing above-mentioned primer to detect G I type G II type norovirus.

Utilize above-mentioned primer to detect the method for G I type G II type norovirus, may further comprise the steps:

1. extracting testing sample RNA, is template with testing sample RNA, in having the RT-PCR reaction system of primer that sequence is Nov-F:5-GTGAATGAWGATGGCGTCKA-3 Nov-R:5-GTHAGWATCCAGGGGTCAAT-3, carries out the RT-PCR reaction;

2. get 5 ml RT-PCR products and in 2% agarose gel electrophoresis that contains the pyridine of bromination second, carry out electrophoresis, whether contain G I type and G II type norovirus nucleic acid in the sample to be checked according to having or not specific band and length to judge behind the electrophoresis.

The present invention adopts the RT-PCR detection method, utilize a pair of norovirus to detect the somatotype universal primer and can realize that disposable processing sample can detect and distinguish norovirus G I type and G II type simultaneously, and the laboratory that is applied to clinical stool sample of success is detected.

Key of the present invention is the design of primer sequence, and the RT-PCR reaction system is formed, reaction conditions is selected and the reaction result judgement all can be undertaken by this area ordinary method.

Said sample RNA extracts and can be undertaken by ordinary method, as adopts RNeasy Mini Kit or other test kit of German QIAGEN company, extracts according to the test kit specification sheets.

The per 25 μ l of said RT-PCR reaction system form as follows:

Figure 2011102975515100002DEST_PATH_IMAGE001

Said RT-PCR reaction conditions is: 50 ℃ of 30min, and 94 ℃ of 3min carry out rt, 94 ℃ of 30s then, 50 ℃ of 30s, 72 ℃ of 1min carry out 35 circulations altogether, 72 ℃ of 10min, 4 ℃ of preservations.

As single positive chain RNA virus, norovirus very easily morphs, and has numerous types.The GI type and the GII type norovirus that wherein cause the human infection can be divided into 14 and 17 genotype at least.Want that the round pcr of using based on nucleic acid amplification detects and distinguish simultaneously G I type and G II type sensitively to norovirus, design one cover is special, the primer of wide spectrum is very crucial.Be applied to the RT-PCT method that norovirus detects in early days, primer design is mostly to being positioned at the RdRp district of ORF1 or the capsid protein district of ORF2.European Union once united 5 different laboratories of 5 countries 5 kinds of conventional RT-PCR methods had been carried out the methodology assessment.The result shows do not have a pair of single primer to reach and detect and distinguish norovirus G I type and G II type simultaneously.The real time fluorescent PCR method that has some to be directed against aforementioned region is again subsequently set up in succession, also all is to be directed against G I type and G II type norovirus design specific primers and probe respectively it is carried out the somatotype detection.

Beneficial effect of the present invention is mainly reflected in: a pair of norovirus Auele Specific Primer of design to the high conservative region of norovirus G I type and G II type capsid protein, detects when being suitable for G I type and G II type norovirus simultaneously; Different (the GI types: 175bp of purpose clip size that utilize this that primer is increased to GI type norovirus and GII type norovirus; GII type: 160bp), can realize that a pair of primer detects and distinguish G I type and G II type norovirus simultaneously at same reaction tubes, not only simplify operation steps, but also can shorten detection time.Experimental result shows that the inventive method has the specificity of height to the detection of norovirus G I and G II type, with other common diarrhea virus rotavirus, EAd, equal no cross reactions of hepatitis A virus of causing; The inventive method can successfully be used for detecting and distinguishing the G I type and the G II type norovirus of actual fecal sample, is highly suitable for the laboratory early diagnosis of the burst epidemic situation that norovirus causes.

Description of drawings

Fig. 1 is the positive strain of known norovirus G I type and G II type, feminine gender and specific detection;

Fig. 2 is the detection of detection method of the present invention to norovirus nucleic acid in the actual sample.

Embodiment

Below in conjunction with accompanying drawing the present invention is done further explain.

This specific embodiment only is to explanation of the present invention; It is not a limitation of the present invention; Those skilled in the art can make the modification that does not have creative contribution to present embodiment as required after reading this specification sheets, but as long as in claim scope of the present invention, all receive the protection of patent law.

Embodiment

1 material and method

1.1 virus

Rotavirus, adenovirus, hepatitis A virus nucleic acid are given Disease Control and Prevention Center's virus institute in Zhejiang Province.The positive strain of known norovirus G I type and G II type is preserved for this laboratory.Clinical samples is that area, Huzhou 2008-2010 non-bacterial acute gastroenteritis is broken out patient's stool sample of gathering in the epidemic situation.

1.2 design of primers

Retrieve 60 strain different year and geographic G I type and G II type norovirus strain complete genome sequence from the GenBank gene pool, carry out homology relatively with biological software, at capsid protein conserved regions design Auele Specific Primer, sequence is:

Nov-F：5-GTGAATGAWGATGGCGTCKA-3 (nt5353-?nt5372)

Nov-R：5-GTHAGWATCCAGGGGTCAAT-3 (nt5528-?nt5510)

Primer entrusts Shanghai Ying Jun Bioisystech Co., Ltd synthetic.G I type and G II type norovirus amplification segment size are respectively 175bp and 160bp.

1.3 RT-PCR reaction:

Norovirus RT-PCR primer sequence,

Nov-F：5-GTGAATGAWGATGGCGTCKA-3 (nt5353-?nt5372)

Nov-R：5-GTHAGWATCCAGGGGTCAAT-3 (nt5528-?nt5510)

Adopt the TAKARA one step RNA PCR kit of company (code:DRR024A), press the test kit specification sheets and operate, reaction system is 25 ml, 10 * RT-PCR damping fluid, 2.5 ml wherein, MgCl ₂5 ml (25mM), dNTP mixture (each 10 mM) 2.5ml, RNase suppressor factor (40U/ml) 0.5 ml; AMV enzyme (5U/ml) 0.5 ml, Taq enzyme (5U/ml) 0.5 ml, each 0.5 ml of the upper reaches and downstream primer (20 μ M); Template ribonucleic acid 5ml, DEPC water 7.5 ml.Reaction conditions is 50 ℃ of 30min, and 94 ℃ of 3min carry out rt, 94 ℃ of 30s then, and 50 ℃ of 30s, 72 ℃ of 1min increase, and change 72 ℃ of 10min, 4 ℃ of preservations after 35 circulations over to.Get 5 ml products and in 2% agarose gel electrophoresis that contains the pyridine of bromination second, carry out electrophoresis, behind the electrophoresis according to have or not specific band (GI:175bp, GII:160bp).Judge and whether contain G I type and G II type norovirus nucleic acid in the sample to be checked.

1.4 RT-PCR specificity test

The RT-PCR reaction system that utilization is set up detects GI type norovirus, GII type norovirus, rotavirus, EAd, hepatitis A virus nucleic acid respectively, verifies its specificity.

1.5 the detection of actual sample

Choose the non-bacterial acute gastroenteritis and break out 101 parts of patient's stool samples gathering in the epidemic situation, detect simultaneously with norovirus RT-PCR method of the present invention and fluorescence RT-PCR method.

2 results

2.1 RT-PCR reaction system and condition

Adopt one step RNA PCR kit (code:DRR024A) the RT-PCR test kit of TAKARA company, the total reaction system is 25 ml.Detect with Eppendorf PCR appearance, reaction parameter is: 50 ℃ of 30min, and 94 ℃ of 3min carry out rt, 94 ℃ of 30s then, 50 ℃ of 30s, 72 ℃ of 1min increase, and change 72 ℃ of 10min, 4 ℃ of preservations after 35 circulations over to.Get 5 ml products and in 2% agarose gel electrophoresis that contains the pyridine of bromination second, carry out electrophoresis, judge behind the electrophoresis to have or not specific band (G I: 175bp, G II: 160bp).

2.2 specificity test

The RT-PCR method that the present invention sets up has specificity preferably to norovirus, to no cross reactions such as other enteroviruses such as rotavirus, EAd, hepatitis A virus.

2.3 the detection of actual sample

Choose the non-bacterial acute gastroenteritis and break out 101 parts of patient's stool samples gathering in the epidemic situation, the primer probe of selecting for use norovirus detection somatotype universal primer of the present invention and 2 covers to be used for the detection of norovirus somatotype respectively carries out RT-PCR and fluorescence RT-PCR simultaneously to sample and detects.The result shows: norovirus RT-PCR method of the present invention detects positive 6 parts of G I altogether, positive 32 parts of G II, negative 63 parts.Adopt fluorescence RT-PCR method detected result to conform to fully with method of the present invention.But the present invention can realize a pair of primer and detect and distinguish G I and G II type norovirus simultaneously at same reaction tubes, not only simplified operation steps, but also can shorten detection time.What Fig. 2 showed is the detection collection of illustrative plates of actual sample.

SEQUENCE?LISTING

< 110>Huzhou City disease prevention and control center

< 120>a kind of primer and utilize this primer to detect the method for G I type G II type norovirus

<130>

<160> 2

<170> PatentIn?version?3.3

<210> 1

<211> 20

<212> DNA

< 213>artificial sequence

<400> 1

gtgaatgawg?atggcgtcka 20

<210> 2

<211> 20

<212> DNA

< 213>artificial sequence

<400> 2

gthagwatcc?aggggtcaat 20

Claims

1. a primer is characterized in that, said primer sequence is following:

Nov-F：5-GTGAATGAWGATGGCGTCKA-3

Nov-R：5-GTHAGWATCCAGGGGTCAAT-3 。