CN108034759A

CN108034759A - A kind of GII type norovirus detection of nucleic acids standard substance and its preparation method and application

Info

Publication number: CN108034759A
Application number: CN201711078576.XA
Authority: CN
Inventors: 徐蕾蕊; 魏海燕; 张西萌; 曾静
Original assignee: Inspection and Quarantine Technology Center Beijing Entry-Exit Inspection and Q
Current assignee: Inspection and Quarantine Technology Center Beijing Entry-Exit Inspection and Q
Priority date: 2017-11-06
Filing date: 2017-11-06
Publication date: 2018-05-15

Abstract

The present invention discloses a kind of GII type norovirus detection of nucleic acids standard substance and its preparation method and application, and GII type norovirus detection of nucleic acids standard substance RNA sequence of the present invention is as shown in SEQ ID No.1.Preparation method the invention further particularly discloses the standard substance and the application in GII type norovirus detection of nucleic acids as standard substance.GII type norovirus detection of nucleic acids standard substance inanimate object infectiousness of the present invention, easily prepares, and has good uniformity and enough stability, and the standard substance has characteristic value that is accurate, can tracing to the source；It can not only be used for the positive control of GII type norovirus nucleic acid qualitative detection, the external standards that can be detected again as GII type norovirus nucleic acid quantification, it can also be used to evaluate new GII type norovirus nucleic acid detection method, material base is provided for GII type norovirus detection of nucleic acids ability certification and accreditation, each laboratory can be widely used in.

Description

A kind of GII type norovirus detection of nucleic acids standard substance and its preparation method and application

Technical field

The present invention relates to technical field of biological.More particularly, to a kind of GII type norovirus detection of nucleic acids standard Material and its preparation method and application.

Background technology

After twentieth century seventies, scholars find in succession NoV, RV, HAstV, adenovirus (Adenoviruses, AdV), letter such as viral (Sapovirus, SaV) etc. is to cause children, the elderly and hypoimmunity crowd's acute watery diarrhea With the main pathogens of severe diarrhea.By taking norovirus as an example, in past more than 10 year, having studied confirms that NoV is to cause urgency Property break out the primary original of causing a disease of nonbacterial gastroenteritis, there is important public health meaning, U.S. CDC study on monitoring is estimated 30%~50% borne Parasitic Encephalopathy gastroenteritis is broken out related with norovirus.At present, the main base of the detection method of food-borne virus In electron microscopic observation, cell culture, nucleic acid hybridization, enzyme linked immunological and PCR (Polymerase Chain Reaction, PCR) etc. technology, but electron microscopic observation, nucleic acid hybridization and enzyme-linked immunoassay method sensitivity it is relatively low, it is impossible to It is used alone and detects, traditional cell culture processes, detection cycle at least needs 7d or so, and many enteron aisle food-borne virus Cytopathic effect is occurred without after cannot carrying out cell culture, or culture, very big puzzlement and challenge are brought to detection work. Therefore, molecular biology method is progressively applied in the detection of food-borne virus in recent years, the time required to shortening detection, Development prospect is brought for the perfect of food-borne virus detection method, standardization and practical application.

PCR as a sensitive Protocols in Molecular Biology, due to the advantage such as easy to operate, quick have become it is food-borne Virus predominantly detects one of technology.Most of food-borne virus are ribonucleic acid (Ribonucleic Acid, RNA) class diseases Poison, so reverse transcription PCR, using more, in order to improve detection sensitivity and specificity, traditional reverse transcriptase polymerase chain formula is anti- (Reverse Transcript Polymerase Chain Reaction, the RT-PCR) technology of answering progressively is improved, increasingly It is perfect, establish shell type, half Nest RT-PCR, multi-primers RT-PCR, can also will be with top the methods of internal standard quantitative RT-PCR Method carries out Multiple Combination, substantially increases detection efficiency and practicality.In recent years, on the basis of RT-PCR reaction systems, hair Put on display Fluorescent quantitative PCR (Real-time Fluorescent Quantitative Polymerase Chain Reaction, qRT-PCR) and digital polymerase chain reaction (Droplet Digital Polymerase Chain Reaction, ddPCR), food-borne virus detection is realized the leap from qualitative to quantitative.

The methods for studying the qRT-PCR for also establishing common food-borne virus in succession many in recent years, as NoV QRT-PCR (Taqman sonde methods) method can specifically detect I type norovirus of G (Norovirus genotype G I, G I) Or II norovirus of G (Norovirus genotype G II, G II), its sensitivity and recall rate compared with conventional RT-PCR method and Enzyme-linked immunosorbent assay (Enzyme-linked Immunosorbent assay, ELISA) method has obtained changing greatly very much It is kind.In the past 20 years, the detection technique of food-borne virus and examination criteria have significant development in food.Wherein, it is international Examination criteria is mainly European Committee for Standardization (European Committee of Standardization, CEN) issue Comite Internationale de Normalisation/technical standard (International Organization for Standardization/ Technical Specifications, ISO/TS) 15216-1 and 15216-2, detection method be qRT-PCR methods；China Coherent detection standard mainly includes：SN/T 4055-2014、SN/T 3841-2014、SN/T 1635-2005、SN/T2520- 2010th, SN/T2519-2010 and SN/T 2518-2010, predominantly RT-PCR, qRT-PCR and link to reverse transcription isothermal duplication (Reverse Transcription Loop-mediated Isothermal Amplification, RT-LAMP) detection side Method.It can be seen from the above that the PCR method based on molecular biology becomes the main means that food-borne virus detects in food.

Although PCR detection techniques continue to develop perfect, its sensitivity and accuracy continuous improvement, in practical operation More influence factor is still had in journey, PCR amplification suppresses after the random error, sample nucleic extraction such as experimenter's operation The remaining of thing, target nucleus acid concentration to be amplified, Reverse Transcription Efficiency etc. can cause testing result deviation.Therefore, it is necessary to using stringent Charge product the reliability that stringent indoor quality control just can guarantee that testing result is carried out to detection process.In addition, quantified Detection, it is necessary to have quantitative standard substance and internal standard.One reliable and stable, and the standard substance of inanimate object infection risk, right It is of great significance in guarantee RNA virus nucleic acid amplification detection quality.As it can be seen that viral nucleic acid augmentation detection standard substance is different Laboratory monitoring testing result comparativity, reagent traceability, the material base of evaluation and the quality control of process of measurement.

It is in the past to use plasmid deoxyribonucleic acid (Deoxyribonucleic Acid, DNA) or contain virion more Positive sample, positive control when being detected such as diarrhea patient fecal specimens as food-borne virus nucleic acid amplification.But Plasmid DNA without Method is controlled the process of viral RNA reverse transcription, for be difficult during quantitative analysis intuitive communicative viral level information.Suffer from Person's fecal specimens have potential infectiousness, and homogeneity is poor, prepare transport difficult, and multigelation restrovirus carrying capacity can substantially drop It is low.The RNA synthesized in vitro can overcome the disadvantages that above-mentioned deficiency, as long as ensureing its enough stability through certain processing, it is possible to as reason The standard items thought carry out quality control to two links of the reverse transcription during food-borne virus detection of nucleic acids and PCR.

In addition, in food the detection of food-borne virus include viral enrichment, viral RNA nucleic acid extraction, RNA reverse transcriptions and Multiple detecting steps such as pcr amplification reaction, wherein virus enrichment and viral RNA extraction process are committed step, it is necessary to it Carry out effective quality control.Pointed out in ISO/TS 15216-1 and ISO/TS 15216-2, food is detected using qRT-PCR When middle hepatitis A virus and norovirus, it is necessary to using can in vitro culture propagation without coating forward direction singlestranded RNA (+ Single-stranded Ribonucleic Acid ,+ssRNA) virus progress quality control of procedure, it is ensured that testing result reliability. The quality control of procedure virus should be similar to detected viral size, but has obvious difference with detected virus gene sequence, And it is not present, can only be artificially added in tested sample in nature.

Therefore it provides a kind of external synthesis GII type norovirus RNA fragment has effectively played in laboratory and laboratory Between quality control action, have broad application prospects.

The content of the invention

First purpose of the present invention be to provide it is a kind of there is excellent homogeneity, sufficiently stable property and can accurately trace to the source The GII type norovirus detection of nucleic acids standard substance of the features such as characteristic value.

Second object of the present invention is to provide a kind of preparation of above-mentioned GII type norovirus detection of nucleic acids standard substance Method.

Third object of the present invention is to provide a kind of above-mentioned GII type norovirus detection of nucleic acids standard substance in GII Application in type norovirus detection of nucleic acids as standard substance.

To reach above-mentioned purpose, the present invention uses following technical proposals：

The present invention provides a kind of GII type norovirus detection of nucleic acids standard substance, the RNA sequence of the standard substance is such as Shown in SEQ ID No.1.

Further, the characteristic value of the standard substance is (5.9 ± 1.2) × 10⁴Copy/μ L.

Invention further provides the preparation method of above-mentioned GII type norovirus detection of nucleic acids standard substance, including with Lower step：

1) GII type norovirus positive is screened, extracts viral RNA, after reverse transcription, is amplified such as SEQ ID No.5 Shown DNA fragmentation；

2) DNA fragmentation described in step 1) is connected construction recombination plasmid with plasmid vector；

3) by the recombinant plasmid transformed that step 2) obtains to competent cell, propagation, extracts recombinant plasmid；

4) recombinant plasmid obtained using restriction enzyme to step 3) extraction carries out single endonuclease digestion, obtains linearisation restructuring Plasmid；

5) to linearize recombinant plasmid as template, in-vitro transcription synthesis obtains GII type norovirus detection of nucleic acids reference material Matter candidate, dilution, after uniformity, Detection of Stability, carries out definite value and uncertainty evaluation, you can.

The GII type norovirus detection of nucleic acids standard substance inanimate object that the present invention is prepared by above method is infectious, keeps away The problem of having exempted from bio-safety, shows through uniformity, Detection of Stability, has good uniformity and enough stability；Through Definite value research and uncertainty evaluation, its characteristic value are (5.9 ± 1.2) × 10⁴Copy/μ L, can accurately trace to the source, and ensure to meet disease Quality Control and quantitative demand during malicious detection of nucleic acids.

Further, the primer such as SEQ ID used in the DNA fragmentation as shown in SEQ ID No.5 are amplified in step 1) Shown in No.2 and SEQ ID No.3.

Further, the restriction enzyme is I restriction enzymes of HamH.

Further, the promoter of the in-vitro transcription is T7 promoters, its sequence is shown in SEQ ID No.6, transcription is Since first G in last three G of sequence.

The plasmid vector and competent cell do not require particularly in the present invention, as long as can meet the need of the present invention Will.In specific embodiment of the present invention, the plasmid vector is pcDNAII carriers；The competent cell is big Enterobacteria TOP10 bacterial strains.

The present invention further additionally provides above-mentioned GII type norovirus detection of nucleic acids standard substance in GII type norovirus Application in detection of nucleic acids as standard substance.Such as can be the positive control of GII type norovirus nucleic acid qualitative detection, External perimysium reference material of GII type norovirus nucleic acid quantification detection etc..

Present invention also offers a kind of kit for including above-mentioned GII type norovirus detection of nucleic acids standard substance.

Further, the kit further includes the RT-ddPCR primer and probes for detecting GII type norovirus nucleic acid Combination, the RT-ddPCR primer and probes combination is comprising the primer as shown in SEQ ID No.2 and SEQ ID No.3 and such as Probe shown in SEQ ID No.4.

Wherein, the probe 5 ' holds flag F AM, 3 ' end mark BHQ.

Beneficial effects of the present invention are as follows：

GII type norovirus detection of nucleic acids standard substance 1 of the present invention) inanimate object infectiousness, easily prepare, have well Uniformity and enough stability, and the standard substance has characteristic value that is accurate, can tracing to the source；2) it can not only be used for GII type promise such as The positive control of viral nucleic acid qualitative detection, and the external standards that can be detected as GII type norovirus nucleic acid quantification, also Available for new GII type norovirus nucleic acid detection method is evaluated, carried for GII type norovirus detection of nucleic acids ability certification and accreditation For material base, each laboratory can be widely used in.

Brief description of the drawings

The embodiment of the present invention is described in further detail below in conjunction with the accompanying drawings.

Fig. 1 shows that GII type norovirus detection of nucleic acids standard substance candidate prepares scheme.

Fig. 2 shows the tem observation result of norovirus positive sample.

Fig. 3 shows the real-time fluorescence RT-PCR testing result of fecal sample norovirus RNA.

Fig. 4 shows pcDNAII Vector maps.

Fig. 5 shows recombinant plasmid structure diagram.

Fig. 6 shows recombinant plasmid regular-PCR qualification result.

Fig. 7 shows linearisation recombinant plasmid size and purity；Wherein, M：DNA Marker；1,2：GII type norovirus line Property recombinant plasmid.

Fig. 8 shows the fluorescent PCR qualification result of in-vitro transcription RNA.

Fig. 9 shows in-vitro transcription RNA sizes and purity；Wherein, M：RNA Marker；1,2：GII type norovirus is external Transcribe RNA.

Figure 10 shows the uniformity of GII type norovirus detection of nucleic acids standard substance.

Figure 11 shows the long-time stability of GII type norovirus detection of nucleic acids standard substance.

Embodiment

In order to illustrate more clearly of the present invention, the present invention is done further with reference to preferred embodiments and drawings It is bright.Similar component is indicated with identical reference numeral in attached drawing.It will be appreciated by those skilled in the art that institute is specific below The content of description is illustrative and be not restrictive, and should not be limited the scope of the invention with this.

Embodiment 1 is used for the specific primer probe for developing GII type norovirus detection of nucleic acids standard substance

Choose II type norovirus Genotyping regional code RdRp's (RNA dependent RNA polymerase) of G Target sequence, sequence analysis and comparison are carried out by NCBI online tools, using Prime Express softwares V4.0 (ABI, Foster City, CA, USA) more than 10 are designed to primer and probe combination, by screening, finally obtain high specificity, be used for Prepare GII type norovirus detection of nucleic acids standard substance and follow-up reverse transcription droplet type digital polymerase chain reaction (Reverse Transcript-droplet digital Polymerase Chain Reaction, RT-ddPCR) detection method primer 1 set each with probe combinations, sequence is shown in Table 1.Its middle probe 5 ' end flag F AM, 3 ' end mark BHQ.Primer and probe is by Beijing The six directions leads to Trade Co., Ltd.'s synthesis.

1 primer probe sequence of table

The preparation of 2 GII type norovirus detection of nucleic acids standard substance candidate of embodiment

The extraction viral RNA from the biological sample (being mostly fecal specimens) for be accredited as the GII type norovirus positive, utilizes The specific primer and probe amplification filtered out goes out the DNA fragmentation as shown in SEQ ID No.5；Then by the DNA fragmentation and matter Grain carrier connection construction recombination plasmid, and by the recombinant plasmid transformed to competent escherichia coli cell, structure contains restructuring The Escherichia coli of plasmid；By the propagation of Escherichia coli, it can be achieved that prepared by a large amount of of recombinant plasmid of above-mentioned DNA fragmentation；Extraction Recombinant plasmid, then carries out single endonuclease digestion to recombinant plasmid using I restriction enzymes of HamH, forms in-vitro transcription template, pass through The synthesis of T7 promoters in-vitro transcription obtains GII type norovirus detection of nucleic acids standard substance candidate.Wherein, the sequence of T7 (TAATACGACTCACTATA (GGG)) as shown in SEQ ID No.6, transcription be since first G in (GGG), because This, obtained GII type norovirus detection of nucleic acids standard substance candidate sequence is as shown in SEQ ID No.1.Specific preparation side In terms of case is shown in Fig. 1, including preparation procedure and Quality Control program two, detailed step is as follows：

1. norovirus positive sample is identified

Norovirus positive excrement is obtained from BJ Children's Hospital, takes 10 μ L fecal specimens drop in the carbon-sprayed copper net of 200 mesh On, inhaled after 1min with filter paper and abandon surplus liquid, 20s is dyed with 2% (w/v) acetic acid uranium, after putting air drying, in transmission electron microscope Lower observation is as it can be seen that have the small round shape structural viral particle being dispersed on a small quantity, diameter is about 30nm or so, meets promise such as in fecal specimens The morphological feature (Fig. 2) of virus.

2. the extraction of norovirus RNA, reverse transcription and identification

The RNA being accredited as with TRIzol extractions in the sample of the norovirus positive, then using SuperscriptTM First-strand synthesis system for RT-PCR kits the reverse transcription reaction for extracting RNA is generated cDNA；The cDNA produced using real time fluorescent PCR method to reverse transcription is identified.It turns out that positive fecal specimens extraction There is specificity fluorescent amplified signal in RNA, and it is 19.9 that it, which detects Ct values, illustrates in the RNA that extracts the G containing higher concentration respectively II type norovirus nucleic acid (Fig. 3).

The preparation of II type norovirus recombinant plasmids of 3.G

The RT-PCR amplifications of 3.1 purpose fragments

Using the primer shown in SEQ ID No.2 and SEQ ID No.3, from the positive fecal specimens of II type norovirus of G In amplify the specific fragment of about 71bp.

Contain 10 × PCR buffer solutions, 5 μ L, 4 μ L of dNTP (2.5mmol/L), sense primer and downstream in 25 μ L reaction systems Each 2 μ L of primer (10 μm of ol/L), 5 μ L of pyrobest enzymes (5U/ μ L) 0.5 μ L and cDNA template.Pressed in ABI 9700PCR instrument The following conditions are reacted：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 45s, 53 DEG C of annealing 45s, 72 DEG C of extension 45s, 35 circulate, 72 DEG C, 8min, 4 DEG C preservations.The recycling of 3.2PCR products

PCR product is usedSV Gel and PCR Clean-Up System (promega) are recycled.PCR is taken to produce 2 μ L of thing carry out 1.5% agarose gel electrophoresis, cut the Agarose plug (as small as possible) containing purpose fragment, are put into 1.5mL centrifugations Guan Zhong, film combination buffer solution is added by 10 μ L/10mg agarose gels, is put 50 DEG C~65 DEG C water-baths and is incubated 10min, is overturned per 2min Mix once, until agarose blob of viscose melts completely.The glue of thawing is moved into adsorption column, places 1min, 16000g at room temperature 1min is centrifuged, the liquid in collecting pipe is discarded, adsorption column is put back in same collecting pipe.It is slow that 700 μ L washings are added in adsorption column Fliud flushing, 16000g centrifugation 1min, discards the liquid in collecting pipe, adsorption column is placed back in collecting pipe, 500 μ L washing buffers Once, 16000g centrifugation 5min, discard the liquid in collecting pipe, adsorption column is put back in collecting pipe, uncapped state to liquid repeated washing Lower centrifugation 1min, to remove remaining ethanol.Adsorption column is put into clean 1.5mL centrifuge tubes, 50 μ L DEPC water are added to Adsorbed film center, stands 1min, room temperature 16000g centrifugation 1min, put -20 DEG C by the liquid eluted (DNA) and save backup. 3.3 coupled reaction

Selection contains carrier of the pcDNAII carriers of T7 promoters as recombinant plasmid, and plasmid map is shown in Fig. 4.First will PcDNAII carriers carry out single endonuclease digestion using EcoRV, form the linearized vector with two flat ends, then expand RT-PCR Increase the purpose fragment to be inserted on pcDNAII carriers by flush end connection.Specific linked system includes 6 μ of PCR glue reclaims product L, 1 μ L of buffer solution, 1 μ L of pcDNAII carriers, 1 μ L of ligase, Yi Shui are connected and supplies reaction system to 10 μ L.It is placed in 14 DEG C of connections Overnight.

RT-PCR is expanded into obtained II type norovirus target gene DNA fragmentations of G and is inserted into carrier EcoRV restriction enzyme sites Place, constructs the recombinant plasmid containing viral target gene, and the structure diagram of recombinant plasmid is shown in Fig. 5.

The conversion of 3.4 connection products

TOP10 competent cells are the competent cells that Escherichia coli TOP10 bacterial strains are handled through special process, can be used Converted in the thermal shock of DNA, be a kind of bacterial strain for being usually used in plasmid cloning.- 80 DEG C of refrigerators take out the TOP10 competent cells of freezing (50 μ L/ pipes) melts on ice, and flicking tube wall makes its mixing.Draw 5 μ L connections liquid add it is above-mentioned fill competent cell from In heart pipe；Flicking pipe outer wall makes its mixing, ice bath 30min；42 DEG C of thermal shock 40s, add 250 μ L SOC nutrient solutions, 37 DEG C of incubations 1h.40 μ L X-gal (20mg/mL) and 4 μ L IPTG (200mg/ are added on the LB tablets containing 50 μ g/mL Ampicillin ML), whole planar surface is spread evenly across with sterile glass spreader, after being dried up on superclean bench, takes 100 μ L bacteria suspensions It is coated on LB tablets, 37 DEG C of culture 12h are stand-by up to growing single bacterium colony.The extraction of 3.5 recombinant plasmids

Aseptic inoculation ring random picking white single bacterium colony on conversion tablet, is inoculated into and contains 50 μ g/mL equipped with 3mL In the test tube of the LB nutrient solutions of Ampicillin, 220rpm, 37 DEG C of shaken cultivations increase bacterium overnight.Using Wizard Plus SV Minipreps DNA Purification System (promega) are extracted, and concrete operations are carried out by kit specification.Most Recombinant plasmid dna is dissolved in 50 μ L TE afterwards, obtains recombinant plasmid pcDNAII-G II, and it is standby in -20 DEG C of preservations.

3.6 the identification of recombinant plasmid

3.6.1PCR identification

The recombinant plasmid extracted using the primer pair shown in SEQ ID No.2 and SEQ ID No.3 carries out PCR amplification, electricity Swimming identification amplified production.Electrophoresis result shows that recombinant plasmid pcDNAII-G II specificity occurs at 71bp after PCR amplification and expands Increase band (see Fig. 6), prompt to contain the target gene fragment of virus in recombinant plasmid, further verified by sequencing.

3.6.2 sequencing identification

The recombinant plasmid that the PCR that learns from else's experience is accredited as the positive send and carries out sequencing mirror in Shanghai Sheng Gong biotechnologys Services Co., Ltd Surely the recombinant plasmid that the PCR that learns from else's experience is accredited as the positive is sent in the progress sequencing identification of Shanghai Sheng Gong biotechnologys Services Co., Ltd.Root According to the structure of the recombinant plasmid shown in Fig. 5, purpose fragment is inserted at II carrier EcoRV restriction enzyme sites of pcDNA, the enzyme of EcoRV Enzyme site is " GAT！ATC ", therefore should include EcoRV restriction enzyme site partial sequences in purpose fragment upstream and downstream；It is in square frame The purpose fragment of insertion, homology point is carried out by the gene order of norovirus in the purpose fragment sequencing result and GenBank Analysis, with the genetic homologies of II type norovirus separation strains of G up to 100%, illustrates that (particular sequence is recombinant plasmid ) in II type norovirus of G containing sequence as shown in SEQ ID No.5 purpose fragment.

4. the preparation of in-vitro transcription template

The linearisation of 4.1 recombinant plasmids

Usually carry out needing to linearize DNA profiling before in-vitro transcription, so that ensure will not be to viral target gene downstream Sequence excessively transcribed.The BamHI restriction enzymes in position and viral target gene downstream are selected to carry out recombinant plasmid single Digestion, produces 5 ' prominent viscous ends, can effectively avoid the transcription to non-target gene after the digestion.

The purifying of the recombinant plasmid of 4.2 linearisations and electroresis appraisal

Efficient in-vitro transcription depends on the transcription templates of high quality, it is desirable to linearizes DNA profiling and is free of RNase, does not mix Other miscellaneous DNA structures and other components to responsive transcription there may be interference.Therefore we select promega companies of the U.S. Wizard DNA Clean-Up System kits purify linearisation DNA, concentrate.The restructuring of linearisation after purification Plasmid judges its size and purity into row agarose gel electrophoresis.

The purpose fragment size being inserted into II-G II of recombinant plasmid pcDNA is 71bp, and II carrier sizes of pcDNA are 2971bp, therefore the size of recombinant plasmid should be 3042bp.1% agarose gel electrophoresis shows linear recombinant plasmid after purification Size and purity meet expection, linearisation is complete, there is no ring-type and super spirial plasmid structure, and without finding DNA degradation, In-vitro transcription template (Fig. 7) can be used as.

5. the preparation of the RNA of the II type norovirus purpose fragment containing G

The in-vitro transcription of 5.1 RNA

With the Ribo MAXTM Large Scale RNA Production system-T7 kits of Promega companies (article No.：P1300 in-vitro transcription (being carried out by kit operational manual)) is carried out.CRNA RNeasy made from in-vitro transcription MiniElute Cleanup kit (being purchased from QIAGEN companies, article No. 74204) are purified, and concrete operations are said according to kit Bright progress.

The identification of 5.2 in-vitro transcription RNA

5.2.1 fluorescent PCR is identified

In-vitro transcription RNA directly carries out fluorescent PCR amplification, whether identification has remaining DNA points without process of reverse-transcription Son；Whether row fluorescent PCR detection again after RNA progress reverse transcriptions, identification transcription RNA are contained into the specific sequence of virus at the same time Row.The RNA of in-vitro transcription directly carries out fluorescent PCR augmentation detection without reverse transcription, does not as a result occur amplified signal, illustrates to turn DNA is free of in record RNA.It will further be detected after RNA reverse transcriptions through fluorescent PCR, specific amplification curve as a result occur, it was demonstrated that The RNA of transcription includes virulent specific sequence (Fig. 8).

5.2.2 ultraviolet spectrophotometry Purity

Light absorption values of the RNA 260nm and 280nm at of UV detector measure in-vitro transcription, calculate A260 with The ratio of A280.Ultraviolet spectrophotometry survey absorbance 260/A280=2.01, show in-vitro transcription product RNA purity compared with It is high.

5.2.3 RNA sequence measures

The RNA of in-vitro transcription is sent to precious bioengineering (Dalian) Co., Ltd and carries out RNA sequencing identifications.II type promises of G are such as Viral nucleic acid standard substance is prepared by the artificial synthesized RNA obtained by in-vitro transcription, it should contains II type norovirus purposes of G Fragment.The structure of recombinant plasmid according to Fig. 5, since the sequence of T7 transcriptons is：TAATACGACTCACTATA (GGG), Transcription is since first G in (GGG), therefore, except II type norovirus of G is examined in in-vitro transcription RNA fragment sequences Survey outside aim sequence, also contain in the T7 promoters partial sequence of aim sequence upstream and target gene downstream from EcoRV digestions Sequence of the site to BamHI restriction enzyme sites.The RNA sequencing results of in-vitro transcription generation are as shown in SEQ ID No.1：

For one section of 163bp's Be II type norovirus detection of nucleic acids purpose fragments of G in RNA fragments, wherein square frame, by the purpose fragment sequencing result with The gene order of norovirus carries out homology analysis in GenBank, and the genetic homology with II type norovirus separation strains of G can It is as a result consistent with the purpose fragment being inserted into II sequencing results of recombinant plasmid pcDNAII-G up to 100%, illustrate in the RNA for preparing The purpose fragment of II type norovirus of corresponding G containing sequence as shown in SEQ ID No.5.

5.2.4 the non denatured agarose gel electrophoresis identifications of 0.7%-2.0%

The purity of one side assistant analysis RNA, on the other hand identifies the integrality of transcription RNA according to the size of RNA fragments. Electrophoresis result finds occur specific band at 100bp-200bp, is consistent with the RNA sequence size drawn is sequenced, and without it There is (Fig. 9) by non-specific transcription product in him.

The GII type norovirus detection of nucleic acids standard substance candidate inanimate object prepared by above scheme is infectious, keeps away The problem of having exempted from bio-safety, Quality Control and quantitative demand when ensureing to meet viral nucleic acid detection.

The foundation of 3 RT-ddPCR methods of embodiment

RT-ddPCR methods are mainly used for uniformity, stability in norovirus detection of nucleic acids standard substance preparation process With definite value research.The optimum detection scope of RT-ddPCR is 20-2000 copies/μ L, using balance weight method, by RNA reference materials Matter carries out gradient dilution, with reference to One-Step RT-ddPCR Kit forProbes kit methods, to the standard of above-mentioned preparation Material carries out RT-ddPCR detections, and used primed probe is with being shown in Table 1, reference reagent cassette method, and concrete operation method is as follows：

1.PCR reaction systems

2 × one-step RT-ddPCR supermix, 10 μ L, 25mM manganese acetate solution 0.8 μ L, forward primer and reverse primer each 1.0-1.8 μ L, fluorescence probe (as shown in SEQ ID No.2 and SEQ ID No.3) (as shown in SEQ ID No.4) 0.5 μ L, RNA template 2.0 μ L, DEPC water polishings to 20 μ L.Primer probe sequence is shown in Table 1.

2. droplet generates

The RT-ddPCR reaction systems of 20 μ L and 65 μ L droplets generation oil are added separately in 8 hole droplet generation cards, put The generation droplet in droplet generation instrument (QX200, Bio-Rad, Pleasanton, CA).

3.PCR reacts

The droplet (40 μ L) of the Water-In-Oil of generation is slowly transferred in 96 orifice plates, aluminium foil heat-sealing film sealer is placed on commonly Amplified reaction, response parameter are carried out in PCR instrument：60℃30min；95℃5min；94 DEG C of 30s, Tm 60s, 40 circulations；98℃ 10min；4℃Hold.Warming and cooling rate≤2.5 DEG C/sec.

4. result judgement

PCR after reaction, after QX100/200droplet reader detections, obtains copying for RNA in 20 μ L reaction systems Shellfish concentration value, the RNA copy concentrations in sample are：The copy of RNA in sample RNA concentration (copy/μ L)=20 μ L reaction systems Concentration (copy/μ L) × 20 μ L ÷, 2 μ L (RNA template sample-addings amount) × extension rate.

The preparation of 4 GII type norovirus detection of nucleic acids standard substance of embodiment

Dilution, uniformity initial survey and the packing of 1.GII type norovirus detection of nucleic acids standard substance candidates

Water is handled by the GII type norovirus detection of nucleic acids standard substance candidate of preparation using the DEPC without RNase It is diluted to 10^-4, its ultraviolet spectrophotometry survey absorbance A₂₆₀/A₂₈₀=2.04, show the GII type norovirus nucleic acid prepared Examination criteria material candidate RNA purity is higher.In order to examine GII type norovirus detection of nucleic acids standard substance candidate to dilute Whether the distribution of RNA molecule is uniform in solution, by top layer, middle level, the different sample points of bottom three in the middle part of conical centrifuge tube 30 μ L RNA solutions are drawn, are detected using fluorescence RT-PCR method, each sampled point repeats detection 5 times, using single factor test The detection Ct values of the more different sampled points of variance analysis method.Uniformity initial survey shows, the GII type norovirus nucleic acid after dilution Examination criteria material candidate RNA molecule is evenly distributed (table 2).GII type norovirus detection of nucleic acids standard substance after dilution The 1.2mL that candidate is distributed into sterile no enzyme has the screw socket cryopreservation tube (Corning) of sealing ring, and every 0.1mL, prepares 200 altogether A sample unit, obtains GII type norovirus detection of nucleic acids standard substance.

2 GII type norovirus detection of nucleic acids standard substance candidate uniformity initial survey result of table

2.GII type norovirus detection of nucleic acids Certified Reference Material Homogeneity is studied

2.1 extracting unit numbers and sampling mode

16 units are extracted out of 200 sample units, for uniformity testing.Sampling mode is stratified random smapling, Specially：According to the initial of packing, mid-early stage, middle and later periods and finishing steps, 200 cell-averages are divided into 4 layers, 50 every layer Unit, unit in every layer is encoded by 1~50 respectively, using the table of random numbers determine every layer extraction sample number, every layer Randomly select 4 units.

2.2 uniform Journal of Sex Research detection methods

Uniformity testing is carried out using RT-ddPCR methods in embodiment 1, every sample repeats detection 3 times.

2.3 uniformity testings assess statistical model

It is first first into trend test between row bottle after obtaining detection data, when trend is not notable between bottle, point Ji it not calculate uniform between bottle Property with bottle in uniformity, using one-way analysis of variance method carry out statistical analysis, calculate uniformity introduce uncertainty

2.4 uniformity testing data results judge

GII type norovirus detection of nucleic acids Certified Reference Material Homogeneity result of study is shown in Figure 10 and table 3, and table 3 lists GII type promise Such as viral nucleic acid examination criteria material homogeneity analysis data.

3 GII type norovirus detection of nucleic acids Certified Reference Material Homogeneity check analysis data (× 10 of table⁴Copy/μ L)

Data can be calculated in table：

The sum of squares of deviations between group

Intra-class variance and

v₁=m-1=15

v₂=N-m=32

Standard deviation between group

Group internal standard deviation

Counting statistics amount F

According to the free degree (15,32) and given significance 0.05, F is checked in by F distributions tables of critical values_α= 1.9922, compared with the F values being calculated F ＜ F_α, it is believed that between group group internal standard deviation between no significant difference, andIn explanation group measured deviation be less than group between measured deviation, droplet type numeral RT-PCR method it is repeated more satisfactory.

Standard deviation between each group of data

Each n_iAll same, in uniform Journal of Sex Research, s_lThe deviation s that as uniformity of standard substance introduces_bb, then formula can letter Turn to：

Therefore the uncertainty u that uniformity introduces_bbFor

u_bb=s_bb=0.18 (× 10⁴Copy/μ L)

3.GII type norovirus detection of nucleic acids standard substance stability studies

3.1 short-term stabilities and long-time stability research approach

Short-term stability research approach：Using 40 DEG C, room temperature (RT, 20~25 DEG C), 4 DEG C and -20 DEG C, investigate norovirus The stability of transportational process under nucleic acid standard substance sample difference traffic condition；The research approach that long-time stability are stablized：By sample Product are continuous at a temperature of being stored in -80 DEG C to investigate at least six moon, and concrete scheme is shown in Table 4.

4 GII type norovirus detection of nucleic acids standard substance stability study scheme of table

The selection of 3.2 stability study detection methods

Stability investigation is carried out using RT-ddPCR methods, each investigation time point randomly selects 3 standard substance samples Article unit, each sample unit repeat detection 3 times, calculate average value.

3.3 stability assessment data results judge

3.3.1 short-term stability result of study

Data are investigated to short-term stability using variance analysis method and carry out statistical check, analyze GII type norovirus core Stability of the sour examination criteria material sample under different preservation conditions, determines most long transport time limit and traffic condition.As a result table The bright standard substance is degraded rapidly in 40 DEG C of environment, only stablizes preserve 3d at room temperature, and 4 DEG C can stablize preservation 14d, and -20 DEG C can Stablize and preserve 28d (table 5).

5 GII type norovirus detection of nucleic acids standard substance short-term stability of table

*：Room temperature refers to temperature between (20~25) DEG C；^#：Compared with the measured value of the 0th day, p ＜ 0.05

3.3.2 long-time stability result of study

Long-time stability result of study is shown in Figure 11, carries out Trend analysis using linear model, observes long-time stability number According to whether having significant change trend, stability of the standard substance within the given holding time is determined, computational stability introduces not Degree of certainty.GII type norovirus detection of nucleic acids standard substance stability data is given in table 6.

6 GII type norovirus detection of nucleic acids standard substance long-time stability of table investigate result

The evaluation of long-time stability is by the characteristic value in different time bioassay standard material, in potential kinetics mechanism In the case of unknown, generally use (classics) linear model, Y=b₀-b₁X depicts the relation of characteristic magnitude and time, formula In：b₀, b₁For regression coefficient, X is the time, and Y is the characteristic value of standard substance.

Slope by 6 Detection of Stability measure data fitting linear equation of table is：

In formula：

The intercept of linear equation is：

The standard deviation of point among straight line：

The standard deviation of slope is：

It is n-2=5 in the free degree, under confidence level p=0.95 (95% level of signifiance), examines slope to be compared with 0 No to have significant difference, the critical value of t-inspection is 2.571, because

|b₁| ＜ t_0.95,5×s(b₁)=2.571 × 0.019=0.049

So slope is not notable, therefore unstability is not observed.At this time, the term of validity of standard substance is 6 months, no The partial uncertainty that stability introduces is u_s=s (b₁) × X=0.019 × 6=0.11 (× 10⁴Copy/μ L)

4.GII type norovirus detection of nucleic acids standard substance combines definite value scheme

GII type norovirus detection of nucleic acids of the pattern to preparation of Duo Jia laboratory cooperation definite values is carried out using RT-ddPCR Standard substance carries out definite value, with the copy concentrations containing GII type norovirus purpose fragment RNA, i.e., contained by every μ L solution RNA copy numbers are as standard value.

4.1 joint definite value schemes：

Choose with authoritative 9 independent laboratories for possessing progress digital pcr detection of certain technology cooperate and determine Value research, four kinds of RM, every kind of each 3 parts of parallel wrapping units are provided to every independent laboratory at random, while are provided specificity and drawn Thing/probe (being shown in Table 1) and normalizing operation program (Standard Operating Procedure, SOP) and result report model This.Each joint definite value laboratory is required received RM Sample storages in -80 DEG C or liquid nitrogen, draws before detection is started Thing/probe is stored in -20 DEG C.

4.2 SOP are summarized

10 times of gradient dilutions are carried out to GII type norovirus detection of nucleic acids standard substance using balance weight method, in 18 μ L The RNA templates after 2.0 μ L dilutions are added in PCR reaction systems, then the 20 μ L PCR reaction systems containing RNA templates are transferred to micro- In drop generation card, droplet generation is stuck in QX100/200 drop generators and generates nearly 20000 droplets, will be micro- with pipettor Drop is gone in 96 orifice plates, and reverse transcription PCR reaction is carried out on regular-PCR instrument.PCR after reaction, is analyzed using QX100/200 Instrument droplet detects whether each droplet has fluorescence signal one by one, obtains the copy concentrations value of RNA in 20 μ L reaction systems, in sample RNA copy concentrations be：The copy concentrations of RNA (are copied in standard substance sample RNA concentration (copy/μ L)=20 μ L reaction systems Shellfish/μ L) × 20 μ L ÷, 2 μ L (RNA template sample-addings amount) × extension rate.Report testing result, and detection method used is carried out Generality describes.

The collection of 4.3 joint definite value test in laboratory data and technical Analysis

Share 9 independent laboratories and participate in the joint definite value research of GII type norovirus detection of nucleic acids standard substance.9 realities Test order of the room by 1#~9#, number consecutively.

After the result report for receiving 9 laboratories, the generality description first to measurement result and detection method used is pressed The following aspects carries out technical Analysis：

(1) definite value laboratory is respectively combined before detection is started, if by received RM Sample storages in -80 DEG C or liquid nitrogen In, primer/probe is stored in -20 DEG C；

(2) whether each RM samples are using balance weight method progress gradient dilution；

(3) add RT-ddPCR reaction systems in whether be 2 μ L dilution after RM samples；

(4) whether detection data are reported as the form of copy concentrations, i.e., per the RNA copy numbers of μ L solution；

(5) it whether there is data reduction mistake.

By technical Analysis, the testing result average value and standard deviation in each data qualifier laboratory are shown in Table 7.

Combine definite value result in 7 Duo Jia laboratories of table

4.3.1 data equally accurate judges

4.3.1.1 method

Using Cork discuss method examine between each laboratory average value whether equally accurate, i.e.,Wherein For the maximum in m laboratory measurements standard deviation,For the standard of the measured value in i-th laboratory in m family laboratory Deviation.

4.3.1.2 result

According to level of significance α=0.05, the laboratory group number m=9 of participation standard substance definite value, most laboratory repeats Pendulous frequency n=9, obtains critical value C (0.05,9,9)=0.2659.Obtained according to Cochran calculation formula：

C ＜ C (0.05,9,9), examining verification, 9 groups of data are equally accurates without suspicious data group.

4.3.2 whether there is outlier judgement

4.3.2.1 method

Statistics inspection is carried out to the average value of 8 groups of II type norovirus detection of nucleic acids standard substance definite values of G with Rod Dixon principle Test, determine whether outlier.

4.3.2.2 result

Each laboratory definite value average value is as follows by arranging from small to large：

f_(0.01,9)=0.672, r₁And r_nRespectively less than f_(0.01,9), examine and judge without outlier.

4.3.3 normal distribution-test

4.3.3.1 method

II type norovirus detection of nucleic acids standard substance definite value result datas of G are examined using the coefficient of skew and coefficient of kurtosis Normality.

4.3.3.2 result

The coefficient of skew；For detecting the asymmetry of data：

Coefficient of kurtosis, is examined for kurtosis：

B=m₄/(m₂)²=2.079

According to confidence level p=0.99 and detection number 8, table look-up to obtain critical value A_{1 (0.01,9)}=1.41, B₁-B₁' it is critical Section is (1.53,3.86), because in 1.36 1.42,1.53 ＜ of ＜, 2.079 ＜ 3.86, therefore II type norovirus nucleic acid of G is examined Survey the equal Normal Distribution of setting examination data of standard substance.

The standard value of 4.4 G, II type norovirus detection of nucleic acids standard substances determines

Complex art analysis judges that rejecting abnormalities value judges with outlier, normal distribution-test and equally accurate are examined, the mark The quasi- equal Normal Distribution of material setting examination data, and the average value of each laboratory definite value result is equally accurate, therefore use Standard value of the overall average of the average value of each laboratory definite value result as the standard substance.I.e. with II type norovirus cores of G 8 groups of statistical averages of sour examination criteria material definite valueAs a row independent measured data, II type norovirus cores of G are calculated The overall average of the quasi- substance characteristics value of acidity scaleStandard deviation s and relative standard deviation.

Calculation formula：(m=9, represents definite value group number)=5.93 (× 10⁴Copy/μ L).

Using with Bessel Formula, standard deviation is calculated, i.e., by the standard deviation of measurement data, pendulous frequency and is wanted The definite value A class uncertainties u that the confidence level asked is calculated by statistical method_A, and calculate opposite A class uncertainties u_A(rel)：

5 GII type norovirus detection of nucleic acids standard substance uncertainty evaluation of embodiment and its characteristic value determine

GII type norovirus detection of nucleic acids standard substance uncertainty source includes three aspects：What inhomogeneities introduced Uncertainty (u_bb), unstability introduce uncertainty (u_s) and definite value introduce uncertainty (u_char).Standard substance Definite value result should have the implication of two aspects：(1) it is tested the standard value y of the best estimate, i.e. standard substance of characteristic magnitude； (2) the uncertainty U of standard value_CRM.Therefore the definite value result of the standard substance is represented by y ± U_CRM。

1. appraisal procedure：

Expanded uncertainty U_CRMIt is that, according to fiducial probability, selection is accordingly after partial uncertainty combined standard uncertainty Spreading factor, using formula U_CRM=k × u_CRMCalculate.u_CRMFor combined standard uncertainty,u_charIt is the not true of standard substance definite value process introducing Fixed degree, by A class components u_AWith B class components u_BTwo parts form, u_bbThe uncertainty introduced for the uniformity of standard substance, u_sFor The uncertainty that the stability of standard substance introduces.Normal distribution formula, Coverage factor k can divide position according to confidence level from t Checked in number.

2. uncertainty evaluation result

Each partial uncertainty is shown in Table 8.

2.1 the uncertainty that uniformity introduces

See that 2.4 uniformity data results judge in the preparation of embodiment 4GII type norovirus detection of nucleic acids standard substances. The uncertainty u that uniformity introduces_bbFor：

u_bb=s_bb=0.18 (× 10⁴Copy/μ L)

Relative uncertainty degree is：

The uncertainty that 2.2 stability introduce

See 3.3.2 Journal of Sex Research knots steady in a long-term in the preparation of embodiment 4GII type norovirus detection of nucleic acids standard substances Fruit.The partial uncertainty that stability introduces is u_s=s (b₁) × X=0.0096 × 6=0.11 (× 10⁴Copy/μ L), relatively It is uncertain

The uncertainty that 2.3 definite values introduce

Uncertainty (the u of definite value_char) mainly by the A class uncertainties (u of Duo Jia laboratories joint definite value result_A) and it is fixed The B class uncertainties (u of value method_B) composition.Due to combining deterministic models using more independent laboratories, in measurement process B classes component is embodied in definite value result at random, therefore no longer carries out B class uncertainty evaluations.Definite value introduce uncertainty be：

Relative uncertainty degree is：

2.4 expanded uncertainty

Each partial uncertainty is synthesized, obtains combined standard uncertainty, takes spreading factor k=2, is calculated each RM's Expanded uncertainty.Expanded uncertainty generally gives up to two effective digitals, and the digit of standard value y will be with uncertainty Digit is alignd after decimal point, after thus making the decimal point of standard value reservation of GII type norovirus detection of nucleic acids standard substance Digit.

8 GII type norovirus detection of nucleic acids standard substance uncertainty (× 10 of table⁴Copy/μ L)

The characteristic value of 2.5 GII type norovirus detection of nucleic acids standard substances

According to standard value and its uncertainty, the characteristic value for determining GII type norovirus detection of nucleic acids standard substance is： 5.9±1.2(×10⁴Copy/μ L).

Obviously, the above embodiment of the present invention is only intended to clearly illustrate example of the present invention, and is not pair The restriction of embodiments of the present invention, for those of ordinary skill in the field, may be used also on the basis of the above description To make other variations or changes in different ways, all embodiments can not be exhaustive here, it is every to belong to this hair Row of the obvious changes or variations that bright technical solution is extended out still in protection scope of the present invention.

Sequence table

<110>Inspection and Quarantine Technology Center, Beijing Entry-Exit Inspection and Q

<120>A kind of II type norovirus detection of nucleic acids standard substances of G and its preparation method and application

<130> JLC17I0607E

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<213>Artificial sequence (Artificial Sequence)

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gggcgaauug ggcccucuag augcaugcuc gagcggccgc cagugugaug gaucccugga 60

gaucauggua aaauuuuccc cagaaccaca ccuggcccag aucguugcag aggaucuccu 120

aucuaucugc agaauuccag cacacuggcg gccguuacua gug 163

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<213>Artificial sequence (Artificial Sequence)

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ccctggagat catggtaaaa tttt 24

<210> 3

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<213>Artificial sequence (Artificial Sequence)

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agataggaga tcctctgcaa cga 23

<210> 4

<211> 21

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 4

cccagaacca cacctggccc a 21

<210> 5

<211> 71

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 5

ccctggagat catggtaaaa ttttccccag aaccacacct ggcccagatc gttgcagagg 60

atctcctatc t 71

<210> 6

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

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taatacgact cactataggg 20

Claims

A kind of 1. GII type norovirus detection of nucleic acids standard substance, it is characterised in that the RNA sequence of the standard substance such as SEQ Shown in ID No.1.
2. GII type norovirus detection of nucleic acids standard substance according to claim 1, it is characterised in that the reference material The characteristic value of matter is (5.9 ± 1.2) × 10⁴Copy/μ L.
3. a kind of preparation method of GII type norovirus detection of nucleic acids standard substance as claimed in claim 1 or 2, its feature It is, comprises the following steps：

1) GII type norovirus positive is screened, extracts viral RNA, after reverse transcription, is amplified as shown in SEQ ID No.5 DNA fragmentation；

2) DNA fragmentation described in step 1) is connected construction recombination plasmid with plasmid vector；

3) by the recombinant plasmid transformed that step 2) obtains to competent cell, propagation, extracts recombinant plasmid；

4) recombinant plasmid obtained using restriction enzyme to step 3) extraction carries out single endonuclease digestion, obtains linearisation restructuring matter Grain；

5) to linearize recombinant plasmid as template, in-vitro transcription synthesis obtains GII type norovirus detection of nucleic acids standard substance and waits Thing is selected, is diluted, after uniformity, Detection of Stability, carries out definite value and uncertainty evaluation, you can.
4. preparation method according to claim 3, it is characterised in that amplified in step 1) as shown in SEQ ID No.5 DNA fragmentation used in primer as shown in SEQ ID No.2 and SEQ ID No.3.
5. preparation method according to claim 3, it is characterised in that the restriction enzyme is restricted interior for HamH I Enzyme cutting.
6. preparation method according to claim 3, it is characterised in that the promoter of the in-vitro transcription is T7 promoters, Its sequence is shown in SEQ ID No.6, transcription is since first G in last three G of sequence.
7. a kind of GII type norovirus detection of nucleic acids standard substance as claimed in claim 1 or 2 is in GII type norovirus core Application in acid detection as standard substance.
A kind of 8. kit of the GII type norovirus detection of nucleic acids standard substance comprising described in claim 1 or 2.
9. kit according to claim 8, it is characterised in that the kit is further included for detecting GII type promise such as The RT-ddPCR primer and probes combination of viral nucleic acid, the RT-ddPCR primer and probes combination include such as SEQ ID No.2 With the primer shown in SEQ ID No.3 and the probe as shown in SEQ ID No.4；Preferably, the probe 5 ' holds flag F AM, 3 ' end mark BHQ.