CN104313187B - GI genome type norovirus reverse transcription Rolling Circle Amplification methods - Google Patents

GI genome type norovirus reverse transcription Rolling Circle Amplification methods Download PDF

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CN104313187B
CN104313187B CN201410632174.XA CN201410632174A CN104313187B CN 104313187 B CN104313187 B CN 104313187B CN 201410632174 A CN201410632174 A CN 201410632174A CN 104313187 B CN104313187 B CN 104313187B
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vibrio
primer
norovirus
type norovirus
reverse transcription
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CN104313187A (en
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黄朱梁
薛超波
王萍亚
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Zhoushan Institute Of Calibration And Testing For Quality And Technology Supervision
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Abstract

The present invention relates to a kind of detection method of I type norovirus reverse transcription rolling circle amplification.This gene chip comprises solid phase carrier and oligonucleotide probe, and described oligonucleotide probe comprises one or more that choose from following nucleotide sequence: the DNA sequence dna 1) chosen from Vibrio hollisae, Vibrio vulnificus, vibrio cholerae, Vibrio parahaemolyticus, Vibrio harveyi, vibrio alginolyticus, Vibrio furnissii, Vibrio mimicus and Vibrio damsela gene; 2) complementary dna sequence of the DNA sequence dna chosen above-mentioned 1); 3) above-mentioned 1) or 2) in the complementary RNA sequence of DNA sequence dna chosen.Described test kit comprises above-mentioned gene chip.Utilize gene chip of the present invention and test kit to detect nine kinds of kinds of pathogenic vibrio in sea-food, easy and simple to handle, sensitivity is good, and repeatability is strong.

Description

GI genome type norovirus reverse transcription Rolling Circle Amplification methods
Technical field
The invention belongs to molecular biology virus detection techniques field, be specifically related to a kind of GI genome type norovirus reverse transcription rolling circle amplification.
Background technology
Zhejiang Province is the large province in ocean, sea-food kind and abundant, and seafood prods are liked by the local common people deeply, and much resident has the custom eating seafood raw.Marine food, especially shellfish, because the feature of its filter-feeding, become the carrier of food-borne virus as hepatitis A virus (HAV), norovirus etc., likely cause the popular of human diseases.The pollution of sea-food virus generally refers to that the viral enrichment of shellfish is polluted, after usually due to water body experience virus contamination after shellfish enrichment, by viral communication to the mankind.Human virus's disease there is no specifics treatment so far, the monitoring of virus is become to the effective means of prevention and corntrol.On the other hand, shellfish exported to me again and again execute inspection norovirus due to external in recent years, erected actual together " green barrier ", within a very long time, we can only deal with external inspection passively.
The people such as norovirus (Norovirus, Nov) belongs to Caliciviridae, Kapikian in 1972 have found this virion, by turning out to be norovirus first by application immuno-electron microscope detection technique in the fecal suspension of this area diarrhoea person.Norovirus is the main pathogen of acute gastroenteritis, can infect the crowd of each age group.According to genetic component type, this virus is divided into three different genus: gene I group, and representative strains is norovirus; Gene II group, representative strains is SnowMountainVirus (SMV); And Sapporo virus.The norovirus analysis of repeatedly Disease code having been confirmed to GI genome type (hereinafter referred to as I type norovirus) and GII genome type is the modal virus strain infecting people in world wide.But I type norovirus is difficult to obtain due to its positive sample, domestic relevant report is also few, virtually adds the difficulty of research.
RT-PCR technology studies the main method that in food, norovirus detects at present.But PCR method is restricted in the application due to False Positive Effect and testing cost costliness; Simultaneously also there is blood many impacts sensitivity and specific factor in PCR method, comprises detected sample itself, the purifying of viral nucleic acid, the DNA fragmentation of pcr amplification be less than 1kb and can not be used for whole genome amplification etc.Therefore, be necessary the detection method setting up a set of I type norovirus, improve the detection and control ability of China's food origin disease.
Summary of the invention
The object of this invention is to provide to a kind of detection method of I type norovirus, thus sensitiveer, the specific I type norovirus detected in fishery products.
First the present invention provides a kind of primer sets for detecting I type norovirus, comprises primer and the junction fragment of reverse transcription amplification primer and locking-type amplification; Primer information is as follows:
Reverse transcriptase primer NVGI-F1:AGTTGGTACCGGAGGTTAATG (SEQIDNO:1)
Reverse transcriptase primer NVGI-R1:GGGATTAAGATGAGGGCCTAAA (SEQIDNO:2)
In addition, the present invention is also provided for the primer improving reverse transcription product concentration, and its information is as follows:
NVGI-F2：CAGTTGGTACCGGAGGTTAATG(SEQIDNO:3)
NVGI-R2：GGGCCTAAACTCAAATCAAACAA(SEQIDNO:4)
The present invention is also provided for the primer and the probe that detect I type norovirus, and its sequence information is as follows:
Primer NVGI-RCAF:CCTCTTGCAATGGATCCTGTAG (SEQIDNO:5)
Primer NVGI-RCAR:CCAAGGGTCAATAGGGTTAACTT (SEQIDNO:6)
Junction fragment NVGI-RCAP:
CAACTGCTGGGCAAGTTAACTCTGACCCTCTTGCAATGGATCCTGTAGCGGGCTCTTCAACAGCAGTTGCAACTGCTGGGCAAGTTAACCCTATTGACCCTTGGGGGCTCTTCAACAGCAGTTG(SEQIDNO:7)。
The present invention also provides a kind of test kit utilizing above-mentioned primer sets to prepare.
The present invention also provides a kind of method utilizing above-mentioned primer sets to detect I type norovirus, includes following steps:
1) extraction of viral RNA to be detected:
The viral RNA that test kit extracts detected sample is extracted with viral RNA;
2) reverse transcription product is obtained with the viral RNA of reverse transcriptase primer NVGI-F1, NVGI-R1 reverse transcription amplification sample;
In order to increase concentration, the reverse transcription product of again increase with NVGI-F2 and NVGI-R2 NVGI-F1, NVGI-R1;
3) rolling circle amplification step: by step 2) reverse transcription product be template, increase with primer sets NVGI-RCAF, NVGI-RCAR and NVGI-RCAP, amplification temperature be 60 DEG C, water bath with thermostatic control reaction 60min, 80 DEG C heating 2min, termination reaction;
4) result detects: amplified production is identified through 1.5% agarose gel electrophoresis, and on sepharose, form stair-stepping band after electrophoresis is positive.
Primer sets of the present invention and method somewhat following:
1. high specificity: method provided by the invention, first a probe (also known as padlock probe) is attached in template, template is made to form ring texture, then adopt a pair Auele Specific Primer, identify 2 sites in circular template, the I type that increases specifically norovirus, and amplified reaction can not be there is to other virogenes; 2. isothermal: reaction carries out under the condition of continuous isothermal, changes temperature without spended time; 3. easy and simple to handle: only need to add various reaction reagent in reaction tubes and just can react, do not need complicated operation steps; 4. detect simple: only need be identified by agarose gel electrophoresis.
Accompanying drawing explanation
Fig. 1: the susceptibility of primer sets of the present invention detects electrophorogram,
Wherein: M:DL2000DNAMarker; The template concentrations (starting point concentration is 27.2ng/ μ L) of swimming lane 1,2,3,4,5,6 is respectively 10 -1, 10 -2, 10 -3, 10 -4, 10 -5, 10 -6; Swimming lane 7 is with aseptic RNaseFreedH 2the blank that O does;
Fig. 2: the specific detection electrophorogram of primer sets of the present invention;
Wherein: M:DL2000DNAMarker; Swimming lane 1:I type norovirus; Swimming lane 2:II type norovirus; Swimming lane 3: new bunyavirus; Swimming lane 4: small round virus; Swimming lane 5: hepatitis A virus (HAV); Swimming lane 6: rotavirus; Swimming lane 7: make volume blank with aseptic RNaseFreedH2O.
Embodiment
Below in conjunction with embodiment, method of the present invention is described in detail.
One, for detecting the design of I type norovirus primer sets
The present invention is according to the gene order of the I type norovirus announced in GeneBank, after tetraploid rice, the specific recognition sequence at padlock probe two ends is designed from highly conserved sequence sequence, closely adjacent after the specific recognition sequence at two ends is combined with single-stranded template, from template, choosing the linkage section part of one section of sequence as padlock probe; The primer of RT-PCR amplification and the primer of rolling circle amplification are by design of primers functional design on NCBI website, and primer is as shown in table 1:
The RT-PCR primer of table 1:I type norovirus and RCA primer
Two, the effect detection of I type norovirus primer sets
1.1 experiment material
Virus strain: I type norovirus, II type norovirus, new bunyavirus
1.2 viral RNAs extract
Get 200 μ L ight soil liquid in the aseptic EP pipe of 1.5mL, extract test kit (QIAGEN) with viral RNA and extract viral RNA, frozen in-70 DEG C, for subsequent use, as the template of RT-PCR amplification.
1.3RT-PCR amplicon virus RNA
Adopt One step RT-PCR test kit (TaKaRa) in conjunction with primer pair NVGI-F1/NVGI-R1 amplicon virus RNA.If one time amplified production concentration is lower, NVGI-F2/NVGI-R2 primer pair can be adopted with an amplified production for template, carry out secondary amplification.It is 27.2ng/ μ L that ultramicron nucleic acid-protein determinator measures production concentration, frozen in-20 DEG C, for subsequent use, as the template of rolling circle amplification.
1.4 rolling circle amplification reaction conditions and systems
1.4.1 ligation
10 μ L ligation systems: 10 × T4DNALigaseBuffer1 μ L, NVGI-RCAP0.1 μM, RT-PCR product 2 μ L, dH 2o5.5 μ L, 94 DEG C of 5min, ice bath 2min, then adds T4DNALigase1U immediately, 37 DEG C of 60min.
25 μ L rolling circle amplification reaction systems: each 0.4 μM of dNTPs0.4 μM, NVGI-RCAF/NVGI-RCAR, BstDNApolymerase (NewEnglandBioLabs) 8U, 10 × reaction buffer 2.5 μ L, above-mentioned connection product 2 μ L, dH 2o complements to 25 μ L, and amplification temperature is 60 DEG C, reacts 60min in thermostat water bath, 80 DEG C of heating 2min, termination reaction.Amplified production is identified through 1.5% agarose gel electrophoresis.With aseptic dH 2o replaces template as negative control.
The sensitivity evaluation of 1.5RCA method
Recording RT-PCR production concentration through ultramicron nucleic acid-protein determinator is 27.2ng/ μ L.First be that the product of 27.2ng/ μ L carries out 10 times of serial dilutions by concentration, then get 2 μ L successively and make template, carry out RCA amplification.With aseptic RNaseFreedH 2o replaces template as negative control.Get 3 μ LRCA amplified productions, through 2% agarose gel electrophoresis qualification.
The Evaluation on specificity of 1.6RCA method
Respectively with I type norovirus, II type norovirus, new bunyavirus, small round virus, hepatitis A virus (HAV), rotavirus nucleic acid for template, carry out specific test by the I type norovirus RCA detection system set up.With aseptic RNaseFreedH 2o replaces template as negative control.
2 results and analysis
2.1 with I type norovirus nucleic acid for template, on sepharose, form stair-stepping band after electrophoresis; The negative control that sterilized water replaces, does not have band after electrophoresis on gel.
2.2 sensitivity technique results
The minimum detectability that the electrophoresis result of Fig. 1 shows this method is 2.72 × 10 -5ng/ μ L.
2.3 specific detection results
By the I type norovirus RCA detection system set up in this test, I type norovirus can be detected, and can't detect non-I type norovirus, as shown in Figure 2.Illustrate that primer has specificity, can be used as the primer that I type norovirus RCA detects.
2.4 shellfish enteraden histologic results
Get shellfish enteraden and organize 10g, shred and stir into homogeneous shape, in enteraden, add the positive ight soil supernatant of 1mLGI type norovirus, vortex mixes.Add 20mL glycine (0.05M)-Nacl (0.15M, pH9.5), violent vortex oscillation 5min, the centrifugal 10min of 5000rpm, sucts and adds chloroform butanols (1:1) in the ratio of 1:1 clearly, vortex oscillation 5min, the centrifugal 10min of 5000rpm, draws supernatant.Supernatant regulates pH7.2 ~ 7.5, adds the PEG6000 of 40%, makes final concentration be 10%; Add the Nacl of 5M, make final concentration be 0.3M.Abundant vortex mixing, is placed in 4 DEG C of refrigerator precipitates overnight, the centrifugal 30min of 10000rpm.Abandon supernatant, the EDPC water of precipitation 1mL is resuspended.Add chloroform butanols (1:1) in the ratio of 1:1 in re-suspension liquid, the centrifugal 5min of thermal agitation 5min, 5000rpm, get supernatant RNA to be carried.
Following step is with embodiment two.Result shows to detect GI type norovirus in shellfish enteraden tissue, and this RT-RCA method of simultaneous verification can detect GI type norovirus.
The above results shows, RCA primer of the present invention and detection method is highly sensitive, high specificity, and the detection be expected to for China I type norovirus nucleic acid provides a kind of sensitivity, special experimental technique.

Claims (5)

1. for detecting primer sets and the probe of I type norovirus, it is characterized in that, described primer sets comprises the primer of reverse transcription amplification primer and locking-type amplification; Wherein the nucleotide sequence of reverse transcriptase primer is SEQIDNO:1-2; Detect primer and the probe of the locking-type amplification of I type norovirus, its nucleotide sequence is respectively SEQIDNO:5, SEQIDNO:6; The nucleotide sequence of probe is SEQIDNO:7.
2. as claimed in claim 1 for detecting primer sets and the probe of I type norovirus, it is characterized in that, also include the primer improving reverse transcription product concentration, its nucleotide sequence is SEQIDNO:3-4.
3. detect a test kit for I type norovirus, it is characterized in that, described test kit includes primer sets according to claim 1 and probe.
4. the test kit detecting I type norovirus as claimed in claim 3, is characterized in that, also include the primer of raising reverse transcription product concentration according to claim 2.
5. the application of the test kit described in claim 3 or 4 in the detection I type norovirus of non-diseases diagnosis, therapeutic purpose.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103649331A (en) * 2011-02-15 2014-03-19 莱卡生物系统纽卡斯尔有限责任公司 Method for localizedin situdetection of MRNA

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KR100957057B1 (en) * 2007-12-03 2010-05-13 래플진(주) Method for Detection of Nucleic Acids by Simultaneous Isothermal Amplification of Nucleic Acids and Signal Probe

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103649331A (en) * 2011-02-15 2014-03-19 莱卡生物系统纽卡斯尔有限责任公司 Method for localizedin situdetection of MRNA

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* Cited by examiner, † Cited by third party
Title
RT—PCR检测实验小鼠自然和实验感染鼠诺如病毒;向丹丹 等;《上海交通大学学报(农业科学版)》;20130630;第31卷(第3期);第63-69页 *
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