CN101671746B - Kit and oligonucleotide sequence for detecting group A rotavirus - Google Patents
Kit and oligonucleotide sequence for detecting group A rotavirus Download PDFInfo
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Abstract
The invention discloses a kit and an oligonucleotide sequence for detecting group A rotavirus, and particularly relates to a group of oligonucleotides for detecting group A rotavirus, which have oligonucleotide sequences shown in sequence tables SEQ ID No.1 to SEQ ID No.5, a kit containing the oligonucleotides and a detection method thereof. During detection, when a large amount of nucleic acid is synthesized, a byproduct, namely magnesium pyrophosphate precipitate is generated, the specificity is extremely high, and whether amplification is carried out or not can be judged only by observing the turbidity of an amplification product by naked eyes.
Description
Technical field
The present invention relates to a kind of test kit and oligonucleotide of the A of detection rotavirus; More particularly; It is a kind of reverse transcription-ring mediated isothermal amplification (RT-LAMP) quick detection kit of the A of detection rotavirus; Particularly be meant oligonucleotide sequence, test kit and the method for utilizing RT-LAMP principle rapid detection A rotavirus, belong to biological technical field.
Background technology
Rotavirus (Rotavirus) belongs to Reoviridae, rotavirus.Spherical in shape, diameter 60～80nm, double capsid, no coating is observed under Electronic Speculum after the negative staining, and viral profile is the wheel shape, so the name rotavirus.Be diplornavirus, about 18550bp is made up of 11 gene fragments, and each fragment contains an open reading frame, encode respectively 6 structural protein (VP1, VP2, VP3, VP4, VP6, VP7) and 5 Nonstructural Proteins (NSP1-NSP5).According to the antigenicity of rotavirus inner capsid VP6, can it be divided into 7 groups (A～G).The A rotavirus is the most common, and is popular in world wide, is the major cause of infantile diarrhea.According to statistics, the A rotavirus can cause 600,000～800,000 death of child every year, accounts for because of acute gastroenteritis inpatient's 30～60% in developing country.
In addition, rotavirus can be propagated through fecal oral route as a kind of important food-borne virus, and the water of pollution and food especially shellfish are the main carriers of propagating.Shellfish is easy to enrichment virus as the filterable ingestion animal.Shellfish culture and the employed purifying method in results back only work to Bacterial Contamination at present, and be invalid to virus.In addition, for keeping mouthfeel fresh and tender, people like edible give birth to or half-mature shellfish.Therefore feed give birth to or without the shellfish of suitably cooking, very easily cause viral diarrhea sick popular with break out, also can the feedwater industry bring enormous economic loss simultaneously.
Therefore; Research and development are to the method for quick of rotavirus; Can be widely used in fields such as clinical diagnosis, health defence, food safety detection and aquaculture,, promote the development of China's aquatic products and food trade all to have great importance the protection people ' s health.
Electron microscopic observation, separation and Culture and enzyme-linked immunoassay method (ELISA) are mainly adopted in the check of rotavirus in the past.Yet because the Electronic Speculum apparatus expensive, detection sensitivity is low, to a great extent limit its application; Isolation cultivation method sensitivity is lower, needs the specific culture condition, and the cycle is long, is inappropriate for the rapid detection of rotavirus; The ELISA method is easy and simple to handle, and is cheap, and sensitivity and specificity religion are high, but its detection depends on the specific anti antigen-antibody reaction, receives the influence of interfering substance in envrionment conditions and the sample easily.
The Protocols in Molecular Biology that with the nucleic acid amplification is the basis has in recent years become the main means that rotavirus detects, but still has different defects.Like RT-PCR, need 4～5 hours usually, detected result need be passed through nucleic acid electrophoresis, ultraviolet visualization, not only causes the crossed contamination to environment easily, and operator are also had certain toxic action.Real-time fluorescence RT-PCR is highly sensitive because of it, high specificity, can be used for detection by quantitative and be favourably welcome; But also plant and instrument and operator's technical qualification are had higher requirement simultaneously; And expense such as agents useful for same and probe is comparatively expensive in the test, and these all are that extensively carrying out of rotavirus detection brought certain difficulty.
(loop-mediated isothermal amplification, LAMP) technology is the nucleic acid amplification method by a kind of novelty of people such as Notomi exploitation in 2000 to ring mediated isothermal amplification.Be characterized in 6～8 zone design 4～6 species-specific primers, utilize a kind of active archaeal dna polymerase of strand displacement (Bst DNApolymerase) that has,, can accomplish nucleic acid amplification reaction in isothermal condition (about 65 ℃) insulation dozens of minutes to target gene.This method does not need processes such as the thermally denature of template, long-time temperature cycle, loaded down with trivial details electrophoresis, ultraviolet visualization, does not also need accurate instrument and expensive detection reagent., visual result simple to operate because of it, highly sensitive, high specificity have been widely used in the detection of pathogenic micro-organism and the diagnosis of communicable disease.China has taken up in the research of the LAMP of pathogenic bacteria detection technique in recent years; Like streptococcus aureus, Salmonellas, monocyte hyperplasia listeria spp and Vibrio parahemolyticus etc. common in Mycobacterium tuberculosis, shigella dysenteriae, large intestine Ai Xi Salmonella, streptococcus pneumoniae, yersinia enterocolitica and the food, obtained significant effect; Be mainly seen in external part report and detect, like RNA viruses such as dna virus such as hepatitis B virus, hepatitis C virus, simplexvirus, adenovirus and influenza virus, sars coronavirus, Measles virus, mumps virus, respiratory syncytial virus, west Nile virus to the LAMP of virus.All do not see the relevant report of utilizing the RT-LAMP technology that rotavirus is detected at present both at home and abroad.
Summary of the invention
The technical problem that the present invention will solve provides the RT-LAMP oligonucleotide that strong, highly sensitive being used to of a group-specific detects the A rotavirus.
Another technical problem that the present invention will solve provides a kind of simple to operate, result A rotavirus RT-LAMP quick detection kit accurately.
For realizing above-mentioned purpose, the present invention adopts following technical scheme:
The present invention provides one group of oligonucleotide that detects the A rotavirus, is made up of to the oligonucleotide of base sequence shown in the sequence table SEQ ID No.5 sequence table SEQ ID No.1; Wherein SEQ ID No.1 is an outside upstream primer, and SEQID No.2 is an outside downstream primer, and SEQ ID No.3 is inboard upstream primer, and SEQ ID No.4 is inboard downstream primer, and SEQ ID No.5 is the ring primer.See table 1 for details.
Table 1A rotavirus RT-LAMP primer sequence
Primer provided by the present invention has the following advantages: (1) efficient and sensible.Research in recent years shows that introducing ring primer (Loop F or Loop B) will help to improve sensitivity, shorten the reaction times.The present invention designs the special ring primer of rotavirus (Loop B), has effectively improved detection sensitivity, and in the time of 60min, amplification efficiency can reach 10
9～10
10One magnitude, the amplification template limit can reach single copy rotavirus nucleic acid.(2) high specificity adopts 5 primers, and 7 different loci of the other rotavirus NSP3 gene of knowing together fully guarantee the specificity of amplified reaction.
The present invention also provides a kind of test kit of the A of detection rotavirus, is made up of following reagent:
(1) TRIZOL lysate: available from Invitrogen company, article No.: 15596-026;
(2) DEPC water: give birth to worker, article No.: D1005 available from Shanghai;
(3) 2 * RT-LAMP reaction solutions: its component is: 2 * ThermoPol damping fluid, 1.6M trimethyl-glycine, 3.6mMdNTPs, 8mM MgSO
4, 0.4 μ M outside upstream primer (F3), 0.4 μ M outside downstream primer (B3), the inboard upstream primer (FIP) of 3.2 μ M, the inboard downstream primer (BIP) of 3.2 μ M, 1.6 μ M encircle primer (LB); Wherein outside upstream primer (F3) is the nucleotide sequence shown in the sequence table SEQ ID No.1; Outside downstream primer (B3) is the nucleotide sequence shown in the sequence table SEQ ID No.2; Inboard upstream primer (FIP) is the nucleotide sequence shown in the sequence table SEQ ID No.3; Inboard downstream primer (BIP) is the nucleotide sequence shown in the sequence table SEQ ID No.4, and ring primer (LB) is the nucleotide sequence shown in the sequence table SEQ ID No.5; Primer entrusts Dalian precious biotechnology ltd synthetic;
(4) Bst archaeal dna polymerase: 8U/ μ L, available from NEB company, article No. M0275L;
(5) AMV ThermoScript II, 10U/ μ L, available from Promega company, article No. M510A;
(6) negative control: saline water;
(7) positive control: the A rotavirus RNA fragment of in-vitro transcription or contain the DNA of A rotavirus target gene;
(8) colour developing liquid: SYBR Green I dyestuff, available from Invitrogen company, article No. S7563;
In wherein said 2 * RT-LAMP reaction solution:
2 * ThermoPol damping fluid is to be formed by 10 * ThermoPol damping fluid (available from NEB company, article No. M0275L) dilution.2 * ThermoPol damping fluid contains 0.2%Triton X-100,20mM (NH
4)
2SO
4, 20mM KCl, 4mM MgSO
440mM Tris-HCl with pH 8.8;
Trimethyl-glycine: available from Sigma company, article No. 107-43-7;
10mM dNTPs: give birth to worker, article No. D0056 available from Shanghai.
The present invention also provides a kind of method of use that detects the rotavirus test kit:
(1) RNA of extraction sample, i.e. template ribonucleic acid: the process for extracting according to embodiment 5 described viral RNAs extracts template ribonucleic acid, also can adopt other conventional molecular biology methods to prepare the RNA or the cDNA of sample;
(2) RT-LAMP reaction system
Table 2RT-LAMP reaction system
(3) reaction conditions: 63 ℃ of isothermal reactions 90～120 minutes, 85 ℃ made enzyme deactivation in 2 minutes, and reaction promptly finishes.
(4) result judges:
Turbidity is observed: reaction result can judge through naked-eye observation reaction product turbidity, white casse positive, clear negative.Also can PCR be managed 12, centrifugal 2 minutes of 000rpm, reacting positive can see white precipitate in the end at pipe.
Colour-change: adding 2.5 μ l colour developing liquid to reaction end-body system is SYBR Green I optical dye, and positive reaction is fluorescent green, and negative reaction then keeps the fluorescent orange of SYBR Green I dyestuff.
Electrophoresis detection: the amplified production of RT-LAMP method is the stem-loop structure DNA of various different lengthss, so positive reaction detects through 2% agarose electrophoresis and be trapezoid-shaped strips, and negative reaction does not then have trapezoidal amplified band to occur, and can be used as aided detection method.
Advantage of the present invention is: (1) isothermal, rationally avoided all inconvenience that particular requirement brought of conventional PCR to temperature cycle.(2) efficient and sensible is designed the special ring primer of A rotavirus (Loop B), further improves detection sensitivity, and in the time of 60min, amplification efficiency can reach 10
9～10
10One magnitude, the amplification template limit can reach single copy A rotavirus nucleic acid.(3) high specificity designs 5 Auele Specific Primers, and 7 different loci of the other rotavirus NSP3 gene of knowing together guarantee the specificity that increases.(4) expense is low, does not need expensive precision instrument and special reagent, only needs a water bath with thermostatic control to get final product.(5) easy and simple to handle, need not carry out the preparatory sex change of double-strandednucleic acid, in a pipe, promptly can accomplish whole detections.(6) detect simply, when nucleic acid is synthetic in a large number, produce by product-magnesium pyrophosphate deposition, high specificity is arranged.Whether the deposition turbidity just can judge amplification as long as detect by an unaided eye.(7) cloning RNA template adds reversed transcriptive enzyme in the reaction system, get final product the efficient amplification of step realization to RNA.
Below in conjunction with Figure of description and embodiment the present invention is described further, all any this areas of doing according to the disclosure of invention be equal to replacement, all belong to protection scope of the present invention.
Description of drawings
Fig. 1 detects the sensitivity analysis result of the test kit of A rotavirus, 1:4.8 * 10
8Copy; 2:4.8 * 10
7Copy; 3:4.8 * 10
6Copy; 4:4.8 * 10
5Copy; 5:4.8 * 10
4Copy; 6:4.8 * 10
3Copy; 7:4.8 * 10
2Copy; 8:4.8 * 10
1Copy; 9:4.8 copy; 10:4.8 * 10
-1Copy; 11: negative control; 12:100bp DNALadder;
Fig. 2 detects the specificity analyses result of the test kit of A rotavirus, 1:DL2000; 2: Astrovirus; 3: HAV; 4:GI type norovirus; 5:GII type norovirus; 6: rotavirus; 7: negative control.
Embodiment
Embodiment 1:RT-LAMP primer design
Choose the highly conserved sequence of A rotavirus NSP3 gene (GenBank accession No.X81436); Utilize the online software of Primer Explorer V4.0 (http://primerexplorer.jp/elamp4.0.0/index.html); The specificity RT-LAMP primer of design A rotavirus, primer is as shown in table 1.Primer Tm value is between 59 ℃ and 62 ℃; Terminal free energy (G) all less than-4cal/mol, can guarantee the sufficiently high stability of primer; GC content is all about 40%; There is not complementary sequence in primer 3 ' end, can effectively prevent the formation of the inner secondary structure of primer.The rotavirus specificity ring primer (Loop B) that the present invention designed helps to improve detection sensitivity, shortens the reaction times.Article 5, primer, 7 different loci of the other rotavirus NSP3 gene of knowing together fully guarantee the specificity of amplified reaction.Primer entrusts Dalian precious biotechnology ltd synthetic, requires PAGE pure or HPLC is pure.
The foundation and the optimization of embodiment 2:A rotavirus RT-LAMP detection architecture
1. method:
1) Mg
2+Concentration:, regulate Mg respectively according to the reaction system preparation reaction mixture of table 2
2+Concentration is to 2mM, 4mM, 6mM, 8mM, 10mM, 12mM, and behind 65 ℃ of insulation 90min, 85 ℃ are reacted the 2min termination reactions.Compare different Mg
2+Concentration is to the influence of amplification efficiency.
2) dNTPs concentration: according to the reaction system preparation reaction mixture of table 2, regulate the dNTPs final concentration respectively to 0mM, 0.2mM, 0.6mM, 1.0mM, 1.4mM, 1.8mM, behind 65 ℃ of insulation 90min, 85 ℃ of reaction 2min termination reactions.Relatively different concns dNTPs is to the influence of amplification efficiency.
3) trimethyl-glycine concentration: according to the reaction system preparation reaction mixture of table 2, and regulate trimethyl-glycine concentration to 0M, 0.4M, 0.8M, 1.2M, behind 65 ℃ of insulation 90min, 85 ℃ of reaction 2min termination reactions.Relatively the different concns trimethyl-glycine is to the influence of amplification efficiency.
4) inside and outside primer concentration ratio: according to the reaction system preparation reaction mixture of table 2, and the primer concentration ratio was respectively 1: 1,1: 2,1: 4,1: 8 inside and outside regulating, behind 65 ℃ of insulation 90min, and 85 ℃ of reaction 2min termination reactions.The influence of relatively more different inside and outside primer concentration comparison amplification efficiencies.
5) reaction times: according to the reaction system preparation reaction mixture of table 2, respectively at 65 ℃ of insulation 15min, 30min, 45min, 60min, 90min, 120min, 85 ℃ of reaction 2min termination reactions.Relatively reaction times length is to the influence of expanding effect.
6) temperature of reaction: according to the reaction system preparation reaction mixture of table 2, behind 55 ℃, 58 ℃, 60 ℃, 63 ℃, 65 ℃, 68 ℃ insulation 90min, 85 ℃ of reaction 2min termination reactions.Relatively the differential responses temperature is to the influence of expanding effect.
2. result: directly the turbidity of the above-mentioned amplified production of visual inspection changes, or in above-mentioned amplified production, adds SYBRGreen I dyestuff, observes colour-change, or carries out 2% agarose gel electrophoresis and detect, and the result finds Mg
2+It is that 0.8M, inside and outside primer concentration ratio reach at 1: 8 o'clock that concentration selects for use 4mM, dNTPs concentration to select 1.8mM, trimethyl-glycine concentration for use, and the gained expanding effect is best when 63 ℃ of reaction 90～120min, so with this foundation and recommendation response condition of forming as test kit.
Embodiment 3: the composition that detects the test kit of A rotavirus
One, the composition of test kit (being stored in-20 ℃)
(1) TRIZOL lysate: available from Invitrogen company, article No.: 15596-026;
(2) DEPC water: give birth to worker, article No.: D1005 available from Shanghai;
(3) 2 * RT-LAMP reaction solutions: its component is: 2 * ThermoPol damping fluid, 1.6M trimethyl-glycine, 3.6mM
DNTPs, 8mM MgSO
4, 0.4 μ M outside upstream primer (F3), 0.4 μ M outside downstream primer (B3), the inboard upstream primer (FIP) of 3.2 μ M, the inboard downstream primer (BIP) of 3.2 μ M, 1.6 μ M encircle primer (LB); Wherein outside upstream primer (F3) is the nucleotide sequence shown in the sequence table SEQ ID No.1; Outside downstream primer (B3) is the nucleotide sequence shown in the sequence table SEQ ID No.2; Inboard upstream primer (FIP) is the nucleotide sequence shown in the sequence table SEQ ID No.3; Inboard downstream primer (BIP) is the nucleotide sequence shown in the sequence table SEQ ID No.4, and ring primer (LB) is the nucleotide sequence shown in the sequence table SEQ ID No.5; Primer entrusts Dalian precious biotechnology ltd synthetic, requires PAGE pure or HPLC is pure;
(4) Bst archaeal dna polymerase: 8U/ μ L, available from NEB company, article No. M0275L;
(5) AMV ThermoScript II, 10U/ μ L, available from Promega company, article No. M510A;
(6) negative control: saline water;
(7) positive control: the A rotavirus RNA fragment of in-vitro transcription or contain the DNA of A rotavirus target gene;
(8) colour developing liquid: SYBR Green I dyestuff, available from Invitrogen company, article No. S7563.
In wherein said 2 * RT-LAMP reaction solution:
2 * ThermoPol damping fluid is to be formed by 10 * ThermoPol damping fluid (available from NEB company, article No. M0275L) dilution.2 * ThermoPol damping fluid contains 0.2%Triton X-100,20mM (NH
4)
2SO
4, 20mM KCl, 4mM MgSO
440mM Tris-HCl with pH 8.8;
Trimethyl-glycine: available from Sigma company, article No. 107-43-7;
10mM dNTPs: give birth to worker, article No. D0056 available from Shanghai.
Two, the preparation of positive reference substance
With A rotavirus RNA (being provided by the CDC virus disease) is template, utilizes primers F 3 and B3, amplifies the specificity segment of about 193bp, is referred to as NSP3 (NSP3 comprises the amplified target gene of RT-LAMP); T-A clones construction recombination plasmid pCR2.1-NSP3 (the pCR2.1-T carrier is available from Invitrogen company); Give birth to worker company in Shanghai and carry out sequencing; And the gene order (accession No.X81436) of A rotavirus among sequencing result and the GenBank carried out homology analysis, guarantee that its homology reaches more than 95%.Extracting and plasmid DNA purification, is template with it, with the Ribo MAX of Promega company
TM(article No.: P1300) carry out in-vitro transcription (being undertaken by the test kit process specifications), the in-vitro transcription product digests with DNase Large Scale RNA Productionsystem-T7 test kit, removes DNA wherein.Utilize RNeasy MiniElute Cleanup kit (available from QIAGEN company, article No. 74204) to be further purified RNA.Ultraviolet spectrophotometer is measured the concentration of DNA or in-vitro transcription RNA, and passes through the copy number of following formula calculation template molecule respectively.The DNA or the in-vitro transcription RNA that will contain A rotavirus target gene are diluted to 1000 copy/μ L, after the packing in-80 ℃ of preservations, as the positive control of test kit.
DNA copy number/μ L={6.022 * 10
23* [5 * 10
-8G/ μ L] * OD
260}/[plasmid molecule base number * 6.58 * 10
2] in-vitro transcription RNA copy number/μ L=[total content (g/ μ L)/(transcribing molecule base number * 340)] * 6.023 * 10
23
Embodiment 4: detect the specificity and the susceptibility test of the test kit of A rotavirus
1. detection sensitivity analysis
(1) method: the positive control plasmid is carried out 10 times of gradient dilutions to single copy/μ L, respectively get 1 μ L, utilize embodiment 3 described test kits to carry out RT-LAMP and detect, reaction system is seen table 2, behind 63 ℃ of incubation 90min, and 85 ℃ of 2min termination reactions.The turbidity of observing each amplified production changes; Perhaps in each amplified production, add SYBR Green I dyestuff 2.5 μ L, observe colour-change; Also can carry out 2% agarose gel electrophoresis and detect, analyze the detected minimum of test kit institute's ability according to the invention.
(2) result: find to utilize test kit according to the invention, in 90min, can detect the positive control dna of single copy.The result sees Fig. 1.
2. detection specificity analysis
(1) material: rotavirus, Astrovirus, HAV, GI type norovirus and GII type norovirus RNA provide by the CDC virus disease.
(2) method: use test kit according to the invention; Common diarrhea virus is comprised that rotavirus, Astrovirus, HAV, GI type norovirus and GII type norovirus RNA carry out the RT-LAMP augmentation detection, observe the generation that this test kit has or not nonspecific reaction.
(3) result: amplified production is detected through turbidity observation, SYBR Green I dyeing, 2% agarose gel electrophoresis; Discovery has only the A rotavirus specific amplification to occur; All the other viruses detect through RT-LAMP and all are negative, and prove that this method has good specificity.The result sees Fig. 2.
Embodiment 5: detect the detection of the test kit of A rotavirus to clinical sample
1. material: infant's diarrhoea stool sample, totally 16 parts are collected by clinical laboratory of children's hospital in the capital.With PBS faecal samples is processed the suspension of 10% (wt/v), 5000g, centrifugal 5min, get supernatant and be stored in-80 ℃ frozen, subsequent use.
2. method:
(1) extraction of viral RNA: utilize traditional Trizol method to extract ight soil RNA, concrete operations are following: 1. in 100 μ L faecal samples, add 1mL Trizol, behind the concussion 30s, room temperature is placed 5min; 2. add 250 μ L chloroform-primary isoamyl alcohol, concuss 30s, room temperature is placed 5min; 4 ℃, 12000g, centrifugal 5min; 3. supernatant is changed in the new centrifuge tube, add the Virahol concuss mixing 30s of 500 μ L, room temperature is placed 5min; 4. 4 ℃, 5000g, centrifugal 5min; 5. carefully remove supernatant, deposition is cleaned back 12000g, 4 ℃, centrifugal 5min (siphon away supernatant as far as possible, centrifuge tube places on the super clean bench deposition is dried up) with 1mL 70% ethanol; 6. add 50 μ L and do not have Rnase water dissolution RNA (better dissolve in order to make viral RNA, put 60 ℃ of heating 10min); 7. the RNA that extracts is subsequent use in-80 ℃ of preservations.
(2) rt: carry out reverse transcription reaction with SuperscriptTM first-strand synthesis system for RT-PCR test kit (available from Invitrogen company, article No. 18080-051), undertaken by this test kit operation instructions.Obtain cDNA and be used for following test.
(3) RT-PCR and real-time fluorescence RT-PCR detect: contain in the RT-PCR reaction system (25 μ L) 10 * PCR damping fluid, 2.5 μ L, dNTP (2.5mmol/L) 2 μ L, 10 μ mol/L upstream primers (5 '-GGCTTTAAAAGAGAGAATTTCCGTCTGG-3 '; Be numbered SEQ ID No.6 in the sequence table) and each 0.5 μ L of downstream primer (5 '-GATCCTGTTGGCCATCC-3 ', be numbered SEQ ID No.7 in the sequence table), Taq enzyme (5U/ μ L) 0.2 μ L and cDNA template 5 μ L.In ABI 9700PCR appearance, react: 94 ℃ of preparatory sex change 5min by following condition; 94 ℃ of sex change 45s, 53 ℃ of annealing 45s, 72 ℃ are extended 45s, 35 circulations, 72 ℃, 8min, 4 ℃ of preservations.After PCR reaction finishes, get 10 μ L products at 1.5% agarose gel electrophoresis, the gel imaging system observations is also taken pictures.
The real-time fluorescence RT-PCR reaction system of 25 μ L comprises: 2 * Master mix (ABI company; Article No. 4304437) 12.5 μ L, 10 μ mol/L upstream primers (5 '-ACCATCTACACATGACCCTC-3 '; Be numbered SEQ ID No.8 in the sequence table) and downstream primer (5 '-GGTCACATAACGCCCC-3 '; Be numbered SEQID No.9 in the sequence table) each 1 μ L, 10 μ mol/L probes (5 '-FAM-ATGAGCACAATAGTTAAAAGCTAACACT-GTCAA-TAMRA-3 '; Be numbered SEQ ID No.10 in the sequence table; 5 ' end mark fluorescent reporter group FAM of said probe sequence, 3 ' end mark cancellation fluorophor TAMRA) 0.1 μ L and cDNA template 5 μ L.In the ABI7000PCR appearance, react: 50 ℃ of 2min, 95 ℃ of 10min by following condition; 95 ℃ of 15s, 60 ℃ of 1min, 40 circulations.
(4) RT-LAMP detects: utilize test kit of the present invention, reaction system (like table 2) and condition (reaction conditions that is provided when using like test kit in the summary of the invention) that 16 parts of fecal sample cDNA are carried out the RT-LAMP augmentation detection.
3. result: utilize RT-LAMP, RT-PCR and real-time fluorescence RT-PCR that the result that 16 parts of fecal samples detect is seen table 3, the recall rate of visible RT-LAMP is suitable with real-time fluorescence RT-PCR, apparently higher than common RT-PCR.And the RT-LAMP method is compared to RT-PCR and real-time fluorescence RT-PCR; Operate simpler; Need not carry out the preparatory sex change of template, long-time temperature cycle; Also do not need expensive detection reagent such as accurate instrument such as fluorescent PCR appearance and probe, visual result is reliable, thereby the carrying out of the field quick detection work of being more convenient for.
Table 3 children faecal samples The selection result
Sequence table
< 110>People's Republic of China Beijing Entry-Exit Inspection and Quarantine Bureau
< 120>a kind of test kit and oligonucleotide sequence that detects the A rotavirus
<130>
<160>10
<170>PatentIn?version?3.3
<210>1
<211>22
<212>DNA
< 213>the synthetic primers F 3
<400>1
tcttcaaaat?gtcatctcac?aa 22
<210>2
<211>22
<212>DNA
< 213>synthetic primer B3
<400>2
cgtctatagc?attaatggga?tt 22
<210>3
<211>44
<212>DNA
< 213>synthetic primers F IP
<400>3
ccttgcaaat?cagcttccaa?ctcagcagaa?tcaaatagca?gatc 44
<210>4
<211>48
<212>DNA
< 213>synthetic primer BIP
<400>4
gtgtcatcag?ttgagtggta?tctaacaatt?gaatttaact?gctgttca 48
<210>5
<211>25
<212>DNA
< 213>synthetic primer LB
<400>5
ggtctatgga?actaccagat?gatgt 25
<210>6
<211>28
<212>DNA
< 213>synthetic primer
<400>6
ggctttaaaa?gagagaattt?ccgtctgg 28
<210>7
<211>17
<212>DNA
< 213>synthetic primer
<400>7
gatcctgttg?gccatcc 17
<210>8
<211>20
<212>DNA
< 213>synthetic primer
<400>8
accatctaca?catgaccctc 20
<210>9
<211>16
<212>DNA
< 213>synthetic primer
<400>9
ggtcacataa?cgcccc 16
<210>10
<211>33
<212>DNA
< 213>synthetic probe
<400>10
atgagcacaa?tagttaaaag?ctaacactgt?caa 33
Claims (2)
1. one group of oligonucleotide that detects the A rotavirus is characterized in that: be made up of to the oligonucleotide of base sequence shown in the sequence table SEQ ID No.5 sequence table SEQ ID No.1; Wherein SEQ ID No.1 is an outside upstream primer, and SEQ ID No.2 is an outside downstream primer, and SEQ ID No.3 is inboard upstream primer, and SEQ ID No.4 is inboard downstream primer, and SEQ ID No.5 is the ring primer.
2. test kit that detects the A rotavirus is characterized in that: be made up of following reagent:
(1) TRIZOL lysate;
(2) DEPC water;
(3) 2 * RT-LAMP reaction solutions: its component is: 2 * ThermoPol damping fluid, 1.6M trimethyl-glycine, 3.6mMdNTPs, 8mM MgSO
4, 0.4 μ M outside upstream primer, 0.4 μ M outside downstream primer, the inboard upstream primer of 3.2 μ M, the inboard downstream primer of 3.2 μ M, 1.6 μ M encircle primer; Outside upstream primer is the nucleotide sequence shown in the sequence table SEQ ID No.1; Outside downstream primer is the nucleotide sequence shown in the sequence table SEQ ID No.2; Inboard upstream primer is the nucleotide sequence shown in the sequence table SEQ ID No.3; Inboard downstream primer is the nucleotide sequence shown in the sequence table SEQ ID No.4, and the ring primer is the nucleotide sequence shown in the sequence table SEQ ID No.5;
(4) Bst archaeal dna polymerase;
(5) AMV ThermoScript II;
(6) negative control: saline water;
(7) positive control: the A rotavirus RNA fragment of in-vitro transcription or contain the DNA of A rotavirus target gene;
(8) colour developing liquid: SYBR Green I dyestuff;
Wherein, in said 2 * RT-LAMP reaction solution, 2 * ThermoPol damping fluid contains 0.2%Triton X-100,20mM (NH
4)
2SO
4, 20mM KCl, 4mM MgSO
440mM Tris-HCl with pH 8.8.
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| CN102776293A (en) * | 2011-05-10 | 2012-11-14 | 北京林业大学 | Method for auxiliary identification of rotavirus and special primers used therein |
| CN102260752B (en) * | 2011-08-25 | 2013-04-10 | 成都新基因格生物科技有限公司 | Composition, kit and method for detecting group A rotavirus |
| CN105734175A (en) * | 2016-04-25 | 2016-07-06 | 广西壮族自治区兽医研究所 | Pig rotavirus reverse transcription loop-mediated isothermal amplification reagent kit and application thereof |
| CN106222300B (en) * | 2016-08-04 | 2019-11-26 | 北京出入境检验检疫局检验检疫技术中心 | A kind of kit and detection method of accurate quantification detection A rotavirus |
| CN108330211A (en) * | 2017-01-18 | 2018-07-27 | 南京美宁康诚生物科技有限公司 | A group rotavirus/astrovirus/intestinal adenovirus nucleic acid multiple fluorescence PCR detection reagent box and application thereof |
| CN107365684B (en) * | 2017-07-14 | 2019-03-01 | 复旦大学 | A kind of paper micro-fluidic chip and its method for extracting nucleic acid and isothermal amplification method |
| CN108085411B (en) * | 2017-11-06 | 2022-01-18 | 北京出入境检验检疫局检验检疫技术中心 | Group A rotavirus nucleic acid detection standard substance and preparation method and application thereof |
| CN108034762A (en) * | 2017-12-21 | 2018-05-15 | 北京卓诚惠生生物科技股份有限公司 | Multiplex PCR detects six kinds of diarrhea virus primed probe groups |
| CN108396076A (en) * | 2018-04-26 | 2018-08-14 | 四川华汉三创生物科技有限公司 | A kind of detection kit of lead to diarrhea pathogenic microorganism |
| CN108546781A (en) * | 2018-04-26 | 2018-09-18 | 四川华汉三创生物科技有限公司 | It is a kind of detection lead to diarrhea pathogenic microorganism Nucleic acid combinations and its application |
| CN108754032B (en) * | 2018-07-19 | 2022-02-18 | 上海速创诊断产品有限公司 | Isothermal nucleic acid amplification system with high specificity and application thereof |
| CN111748552A (en) * | 2019-12-24 | 2020-10-09 | 深圳市人民医院 | Kit for detecting five infant diarrhea disease series viruses and application thereof |
| CN114854734B (en) * | 2022-04-13 | 2023-11-21 | 福建省农业科学院畜牧兽医研究所 | Pigeon rotavirus A-type loop-mediated isothermal amplification detection primer set and kit thereof |
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