CN103014180A - Detection primer, probe and detection method of human astrovirus nucleotide - Google Patents

Detection primer, probe and detection method of human astrovirus nucleotide Download PDF

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Publication number
CN103014180A
CN103014180A CN2012105843781A CN201210584378A CN103014180A CN 103014180 A CN103014180 A CN 103014180A CN 2012105843781 A CN2012105843781 A CN 2012105843781A CN 201210584378 A CN201210584378 A CN 201210584378A CN 103014180 A CN103014180 A CN 103014180A
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probe
primer
detection
astrovirus
nucleotide
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CN2012105843781A
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肖性龙
余以刚
吴晖
李晓凤
唐语谦
袁琨
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South China University of Technology SCUT
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South China University of Technology SCUT
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Abstract

The invention discloses a detection primer, a probe and a detection method of human astrovirus nucleotide, belonging to the technical field of biological detection. The detection primer of the human astrovirus nucleotide comprises a forward primer and a reverse primer, the nucleotide sequences of which are shown as SEQ NO.1 and SEQ NO.2. A nucleotide sequence of the probe which is matched with the detection primer is shown as SEQ NO.3; and one end of the probe is signed with a report fluorescent dye, and the other end of the probe is signed with a quenching fluorescent dye. The detection method of the human astrovirus nucleotide comprises the following steps of: carrying out real-time fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) amplification by taking a sample RNA (Ribonucleic Acid) to be detected as a template and utilizing the forward primer, the reverse primer and the probe, collecting data after every one circulation is finished, and judging a result according to an amplification curve after the reaction is finished. The primer designed according to a human astrovirus genomic sequence is good in specificity and is high in sensitivity when being used for real-time fluorescent RT-PCR detection.

Description

A kind of detection primer of people's Astrovirus Nucleotide and probe and detection method
Technical field
The present invention relates to technical field of biological, particularly a kind of detection primer of people's Astrovirus Nucleotide and probe and detection method.
Background technology
People's Astrovirus (Human astrovirus, HAstV) is nonencapsulated single strand plus RNA virus, belongs to the Astroviridae Mammals and belongs to.HAstV causes youngling to produce a kind of important pathogen of diarrhoea, all has the report that Astrovirus infects all over the world, both can distribute also to cause outbreak of epidemic, also can cause nosocomial infection.Similar to rotavirus, Astrovirus infect mostly occur 2 years old with interior especially 1 years old with interior infant.Think that at present people's Astrovirus is the second cause of disease of infant's viral gastroenteritis, is only second to rotavirus.In addition, the elderly and immune deficiency patient also are the high risk population that Astrovirus infects, and the human immune deficiency patient, accept bone marrow transplantation and severe combined immunodeficiency patient and Astrovirus occurs infect and all have been reported.People's Astrovirus also is one of cause of disease of normal adults generation gastro-enteritis.Therefore, high specificity is badly in need of in Disease Prevention and Control Institutions and hospital, and susceptibility is high, and is easy to operate, can be used for the method for the Rapid﹠Early diagnosis of the epidemic outbreaks such as gastro-enteritis and clinical case.
Madeley observed Astrovirus first under Electronic Speculum in 1975, because there are 5～6 starlike projections on the virion surface, so gain the name.People's Astrovirus particle diameter is 28～41nm, and when virus is present under the high pH value environment, it shows as typical starlike form.Its genome comprises 5 ' non-coding region, 3 open reading frames (ORF1a, ORF1b, ORF2), 3 ' non-coding region of 1 about 80 Nucleotide and the poly-A tail of 1 about 30 Nucleotide of about 85 Nucleotide to 3 ' end from 5 ' end.ORF1a and ORF1b coding Nonstructural Protein, ORF1a encoding serine proteolytic enzyme, and there is 1 nuclear localization signal in its downstream; The RNA polymerase (RNA-dependent RNA polymerase, RDRP) that the ORF1b coding RNA relies on, RDRP is by 1 synthetic fusion rotein of rrna reading frame shift mechanism.The HAstV capsid protein is synthetic by the ORF2 coding.In the process of virus replication, can detect the RNA of 2 kinds of virus-specifics in the cell that infects, 1 is the full length genomic rna of 6.8KB, and another is the subgenomic RNA of 2.8Kb, and the 3 ' end of 2 kinds of RNA all has poly-A tail.Subgenomic RNA has comprised whole ORF2 sequences, can be used as the mrna expression capsid protein.The ORF2 coded product comprises 2 zones at least: the 1st zone (1～415 amino acid) is high conservative between each serotype; The 2nd zone (416 amino acid～end) be highly the variation, virus in and antigenic determinant namely in this zone.
People's Astrovirus is bred difficulty in cell cultures, and most Astrovirus do not produce cytopathy in cell culture, and this separates for virus and has brought difficulty.At present the conventional sense method of people's Astrovirus mainly is the virion of using in the Electronic Speculum direct-detection excrement, or detects virus antigen in the excrement and the specific antibody in the serum with immune diagnostic method.Electron microscopic examination moroxydine sensitivity is lower, occurs easily undetected; The immunological method specificity has much room for improvement, and all wastes time and energy.Traditional RT-PCR also is used to people's Astrovirus, but need 6 hours detection time.
Summary of the invention
The shortcoming that primary and foremost purpose of the present invention is to overcome prior art provides a kind of detection primer of people's Astrovirus Nucleotide with not enough.
Another object of the present invention is to provide the probe that is used with above-mentioned detection primer.
Another object of the present invention is to provide a kind of method of the people's of detection Astrovirus Nucleotide, the method detects by using above-mentioned detection primer and probe to carry out real-time fluorescence RT-PCR, can be fast simply judgement sample whether people's Astrovirus is arranged, for the etiological diagnosis of people's diarrhoea and gastro-enteritis provides scientific basis.
Purpose of the present invention is achieved through the following technical solutions:
A kind of detection primer of people's Astrovirus Nucleotide, described detection primer is comprised of forward primer and reverse primer, and its nucleotide sequence is as follows:
Forward primer: 5 '-GCCAGACTCACAGAAGAGCAAC-3 ';
Reverse primer: 5 '-GGACTTGCTAGCCATCACACTTC-3 '.
The nucleotide sequence of the probe that is used with above-mentioned detection primer is as follows:
Probe: 5 '-CCTCCCCTCCAAATGCGATGGAG-3 ', this probe one end is marked with the report fluorescence dye, and the other end is marked with the cancellation fluorescence dye; Preferably, 5 ' end of this probe is marked with the report fluorescence dye, and 3 ' end is marked with the cancellation fluorescence dye.
Described report fluorescence dye is preferably Fam, Hex, Tet, Joe, Vic, FITC, Cy3 or Cy5.
Described cancellation fluorescence dye is preferably Tamra, Rox, Dabcy1, BHQ1 or BHQ2.
According to the TaqMan technology, added a nucleotide probe that is marked with two fluorescence dye groups on the basis of conventional PCR, for example will report 5 ' end of the probe of fluorochrome label, the cancellation fluorochrome label is at 3 ' end of probe, both consist of the energy transfer organization, report that namely the fluorescence that fluorescence dye is launched can be absorbed by the cancellation fluorescence dye, when the two is far away apart from change, restraining effect weakens, and report fluorescence dye signal strengthens.In the amplified reaction process, probe is hybridized with the purpose amplified fragments on the template, because the Taq enzyme has 5 ' end to the 5 prime excision enzyme activity of 3 ' end, in the amplification extension stage probe is cut off, the restraining effect of cancellation fluorescence dye disappears, and report fluorescence dye signal strengthens, in the amplification extension stage probe is cut off, the restraining effect of cancellation fluorescence dye disappears, and report fluorescence dye signal strengthens, thereby realizes the detection to people's Astrovirus.
A kind of method that detects people's Astrovirus Nucleotide, comprise the steps: take sample RNA to be checked as template, utilize above-mentioned forward primer, reverse primer and probe to carry out the real-time fluorescence RT-PCR amplification, each loop ends image data, reaction finishes rear according to the amplification curve result of determination.
The real-time fluorescence RT-PCR reaction system of the method for described detection people Astrovirus Nucleotide comprises following component: 2.5 μ L10 * One Step RNA PCR Buffer, 2 each 2.5mM of μ L dNTP(), each 1.5 μ L of above-mentioned forward primer and reverse primer (10 μ M), 0.5 the above-mentioned probe of μ L (10 μ M), 0.5 μ L Taq enzyme, 0.5 μ L RNase Inhibitor(40U/ μ L), 0.5 μ L AMV RTase XL(5U/ μ L), 5 μ L RNA, 10.5 μ L RNase Free dH 2O.
Certainly, the primer that can provide according to the present invention to or the probe of its other type of amplified production sequences Design, detect with the fluorescent PCR of applicable different methods.
The present invention has following advantage and effect with respect to prior art:
(1) the present invention is good according to the primer specificity of people's Astrovirus genome sequence design, is used for the highly sensitive of real-time fluorescence RT-PCR detection.
(2) detection method accuracy of the present invention is high, and is highly sensitive, its can be fast simply judgement sample whether people's Astrovirus is arranged, for etiological diagnosis and the differential diagnosis of people's diarrhoea, gastro-enteritis provides scientific basis.
Description of drawings
Fig. 1 is the specific test figure as a result of people's Astrovirus nucleic acid Real-time RT-PCR detection method.
Fig. 2 is the susceptibility test-results figure of people's Astrovirus nucleic acid Real-time RT-PCR detection method.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited to this.
The design of embodiment 1 primer and probe
Download all known person Astrovirus whole genome sequences from ncbi database, and carry out comparative analysis, select the section without secondary structure and high conservative, design primer and probe, its sequence is as follows:
Forward primer: 5 '-GCCAGACTCACAGAAGAGCAAC-3 ';
Reverse primer: 5 '-GGACTTGCTAGCCATCACACTTC-3 '.
The sequence of probe is: 5 '-CCTCCCCTCCAAATGCGATGGAG-3 '; 5 ' end of this probe is with reporting fluorescence dye VIC mark, 3 ' end cancellation fluorescence dye BHQ2 mark.
The optimization of embodiment 2 people's Astrovirus nucleic acid fluorescent RT-PCR conditions
Utilize people's Astrovirus of deactivation as sample to be checked, extract test kit with commercial RNA and extract virus genome RNA, be stored in after the packing respectively-20 ℃ for subsequent use.
(1) optimization of primer concentration: in the situation that other condition is identical in reaction system, primer concentration among the embodiment 1 is done the multiple proportions serial dilution from 0.1 μ mol/L to 1.6 μ mol/L respectively, analysis by test-results is compared, and determines that best primer final concentration is 0.4 μ mol/L.
(2) optimization of magnesium ion concentration: in the situation that other condition is identical in reaction system, with MgCl 2Concentration increase progressively with 1mmol/L from 1mmol/L to 10mmol/L, determine that the best magnesium ion concentration of magnesium ion is 5mmol/L.
(3) optimization of ThermoScript II (AMV RnaseXL) consumption: use the test-results of different concns ThermoScript II relatively, selected 5U is as the consumption of ThermoScript II.
(4) optimization of Taq archaeal dna polymerase (Taq enzyme) consumption: by comparing the optimization experiment result of Taq enzyme dosage, selected 5U is as the consumption of Taq enzyme.
(5) optimization of dNTPs concentration: detect by the dNTPs that uses different concns, select 1mmol/L as the usage quantity of dNTPs after the comprehensive assessment.
(6) optimization of concentration and probe concentration: in the situation that other condition is identical in reaction system, concentration and probe concentration among the embodiment 1 is done to detect after the multiple proportions serial dilution from 0.1 μ mol/L to 0.5 μ mol/L respectively, analysis by test-results is compared, and determines that best probe final concentration is 0.2 μ mol/L.
Utilize primer and the probe of embodiment 1 to carry out the foundation of reaction system, determine that at last the people's Astrovirus real-time fluorescence RT-PCR reaction system that adopts is 25 μ l systems, required each component and respective concentration see Table 1.
Each component situation in the reaction of table 1 people Astrovirus real-time fluorescence RT-PCR
Component Consumption/final concentration
10×One?Step?RNA?PCR?Buffer 1×
25mmol/L?MgCl 2 5mmol/L
dNTPs 1mmol/L
RNase?Inhibitor 40Unit
Primer 0.4μmol/L?each
Probe 0.2μmol/L
Template 10μl
AMV?RNaseXL 5Unit
Taq 5Unit
Annotate: the instrument that a. uses is different, reaction parameter should be appropriately adjusted.
B. different according to detecting the sample source, should suitably adjust the template dosage.
Q fluorescence RT-PCR condition is selected as follows:
50 ℃ 30 minutes, 95 ℃ 2 minutes, 1 circulation; 95 ℃ 5 seconds, 60 ℃ 40 seconds, 40 circulations.
The foundation of embodiment 3 people's Astrovirus nucleic acid real-time fluorescent RT-PCR method for detecting
(1) extracting of the preparation of template to be measured: viral sample RNA is extracted with Roche High Pure viral RNA kit test kit.
1) gets 0.5 gram stool sample, add the suspension that TE500 μ L makes 10-20%, centrifugal 5 minutes of 8000rpm;
2) get on the 200 μ L samples and reset and add 400 μ L in conjunction with liquid, mix, add in the purifying strainer tube 1000rpm, 15s;
3) discard filtered liquid, change a new collection tube, add 500 μ L inhibitor removal buffer, centrifugal 1 minute of 10000rpm;
4) discard filtered liquid, change a new collection tube, add 450 μ L washing lotions, centrifugal 1 minute of 10000rpm;
5) heavily wash once; Centrifugal at last, 13000rpm, 10s discards remaining washing lotion;
6) remove collection tube, change a clean 1.5mL Eppendrof centrifuge tube without RNA, add in the strainer tube with 50 μ L elutriants, centrifugal 1 minute of 10000rpm moves on to the RNA that extracts in the new centrifuge tube, in-80 ℃ of preservations.
(2) amplification condition of people's Astrovirus real-time fluorescence RT-PCR reaction
According to table 1 application of sample, will add excellent PCR pipe and be placed in the fluorescent PCR instrument, after being set, corresponding phosphor collection condition increases, and response procedures is as follows:
1) 50 ℃ were carried out the reverse transcription of RNA in 30 minutes, 95 ℃ of 3 minutes deactivation ThermoScript II;
2) 95 ℃ of sex change in 15 seconds, 55 ℃ of annealing in 30 seconds, 72 ℃ of extensions in 1 minute so repeat 5 and loop pre-amplification;
3) 95 ℃ of sex change in 10 seconds, are extended at 60 ℃ of annealing in 40 seconds, so repeat 40 augmentation detection that loop the purpose fragment, and test-results can Real-Time Monitoring.
(3) detected result of people's Astrovirus real-time fluorescence RT-PCR
After testing, then show positive amplification curve if contain people's Astrovirus in the sample to be checked, its detection sensitivity can reach 50 copy/mL; If do not contain people's Astrovirus in the nutrient solution to be checked then without amplified signal.
The specific test of embodiment 4 people's Astrovirus nucleic acid Real-time RT-PCR detection methods
The detection method that embodiment 3 is set up in utilization detects (HAstV virus 3 strains to the different human virus of 10 strains, gray nucleus virus Polio I, Polio II, each 1 strain of Polio III, rotavirus 3 strains, norovirus 1 strain), detected result as shown in Figure 1,3 strain HAstV virus (is numbered HAstV-SZ11, HAstV-SZ12, HAstV-SZ13) corresponding specificity fluorescent amplification curve (referring to A indication among the figure) all appears, is positive.And other 7 strain human virus (the gray nucleus virus Polio I, Polio II, the Polio III that comprise 3 strain deactivations, 3 strain rotaviruss, 1 strain norovirus.) then all do not have fluorescent signal to produce (referring to B indication among the figure), be judged to feminine gender.The result shows that designed primer probe is only special to purpose virus (that is, HAstV virus), with other detected object no cross reaction.
Take the RNA of people's Astrovirus as template, detect by real-time fluorescence RT-PCR and can be observed obvious fluorescence intensity and change, the result is all positive.And other viral fluorescence intensity does not change, and the result is all negative.Show that this detection method has good specificity.
The susceptibility test of embodiment 5 people's Astrovirus nucleic acid Real-time RT-PCR detection methods
The RNA of HAstV virus is diluted respectively with the water that DEPC processes, carry out relative sensitivity and detect, in 25 μ L reaction systems, RNA is diluted into respectively 5.0 * 10 7, 5.0 * 10 6, 5.0 * 10 5, 5.0 * 10 4, 5.0 * 10 3, 5.0 * 10 2, 5.0 * 10 1Copy number, detected result as shown in Figure 2, the lowest detectable limit that detects of confirmer's Astrovirus all reaches 50 templates copies as a result.
Above-described embodiment is the better embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.

Claims (6)

1. the detection primer of people's Astrovirus Nucleotide, it is characterized in that: described detection primer is comprised of forward primer and reverse primer, and its nucleotide sequence is as follows:
Forward primer: 5 '-GCCAGACTCACAGAAGAGCAAC-3 ';
Reverse primer: 5 '-GGACTTGCTAGCCATCACACTTC-3 '.
2. the probe that is used with detection primer claimed in claim 1, it is characterized in that: the nucleotide sequence of described probe is as follows:: 5 '-CCTCCCCTCCAAATGCGATGGAG-3 '; 5 ' end of this probe is marked with the report fluorescence dye, and 3 ' end is marked with the cancellation fluorescence dye.
3. probe according to claim 2 is characterized in that: 5 ' end of described probe is marked with the report fluorescence dye, and 3 ' end is marked with the cancellation fluorescence dye.
4. probe according to claim 2 is characterized in that:
Described report fluorescence dye is preferably Fam, Hex, Tet, Joe, Vic, FITC, Cy3 or Cy5;
Described cancellation fluorescence dye is preferably Tamra, Rox, Dabcy1, BHQ1 or BHQ2.
5. method that detects people's Astrovirus Nucleotide, it is characterized in that comprising the steps: take sample RNA to be checked as template, utilize above-mentioned forward primer, reverse primer and probe to carry out the real-time fluorescence RT-PCR amplification, each loop ends image data, reaction finish rear according to the amplification curve result of determination.
6. method according to claim 5, it is characterized in that: described real-time fluorescence RT-PCR reaction system comprises following component: 2.5 μ L10 * One Step RNA PCR Buffer, 2 each 2.5mM of μ L dNTP(), each 1.5 μ L of above-mentioned forward primer and reverse primer (10 μ M), 0.5 the above-mentioned probe of μ L (10 μ M), 0.5 μ LTaq enzyme, 0.5 μ LRNase Inhibitor(40U/ μ L), 0.5 μ L AMV RTase XL(5U/ μ L), 5 μ L RNA, 10.5 μ L RNase Free dH 2O.
CN2012105843781A 2012-12-28 2012-12-28 Detection primer, probe and detection method of human astrovirus nucleotide Pending CN103014180A (en)

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CN103436638A (en) * 2013-09-02 2013-12-11 湖北朗德医疗科技有限公司 Real-time fluorescence RT-PCR (reverse transcription-polymerase chain reaction) detection kit for human astrovirus and application thereof
CN107164562A (en) * 2017-06-27 2017-09-15 华东医院 Correlated virus multiple gene detection architecture of suffering from diarrhoea and its kit and application
CN108085412A (en) * 2017-11-06 2018-05-29 北京出入境检验检疫局检验检疫技术中心 A kind of human astrovirus 1's type detection of nucleic acids standard substance and its preparation method and application

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Cited By (4)

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CN103436638A (en) * 2013-09-02 2013-12-11 湖北朗德医疗科技有限公司 Real-time fluorescence RT-PCR (reverse transcription-polymerase chain reaction) detection kit for human astrovirus and application thereof
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CN107164562A (en) * 2017-06-27 2017-09-15 华东医院 Correlated virus multiple gene detection architecture of suffering from diarrhoea and its kit and application
CN108085412A (en) * 2017-11-06 2018-05-29 北京出入境检验检疫局检验检疫技术中心 A kind of human astrovirus 1's type detection of nucleic acids standard substance and its preparation method and application

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