CN105177180A - Real-time fluorescent quantitative RT-PCR kit for detecting hearstlands virus and real-time fluorescent quantitative RT-PCR detection method for detecting hearstlands virus - Google Patents
Real-time fluorescent quantitative RT-PCR kit for detecting hearstlands virus and real-time fluorescent quantitative RT-PCR detection method for detecting hearstlands virus Download PDFInfo
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Abstract
The invention relates to a real-time fluorescent quantitative RT-PCR kit for detecting hearstlands virus and a detection method for detecting hearstlands virus and belongs to the field of biotechnology. The kit comprises a 2*RT-PCR reacting liquid, a 25*RT-PCR enzyme mixed solution, a forward primer, a backward primer, a probe, a negative control and a positive control, wherein the nucleotide sequence of the forward primer is as show in the sequence 1 of a sequence table; the nucleotide sequence of the backward primer is as shown in the sequence 2 of the sequence table; the nucleotide sequence of the probe is as shown in the sequence 3 of the sequence table, and a 5' end of the probe is connected with a fluorophore, and a 3' end of the probe is connected with a quencher; the negative control is nuclease-free sterile ultrapure water; and the positive control contains a gene of which the nucleotide sequence is as shown in the sequence 4 of the sequence table. The real-time fluorescent quantitative PCR detection kit is convenient to use, is low in consumption of a reagent, greatly simplifies the operation process, reduces pollution in the operation process, and is strong in detection specificity, high in sensitivity and suitable for quickly detecting hearstlands virus.
Description
Technical field
The invention belongs to biological technical field, being specifically related to a kind of real-time fluorescence quantitative RT-PCR (reverse transcription PCR) detection kit for detecting innerland virus and method.
Background technology
Innerland virus (HearstlandsVirus) belongs to bunyaviridae innerland virus group membership, is assigned to recently in Phlebovirus.When this virus was in hospital because of having a fever and having a headache two Missourian citizens in 2012 and is gone to a doctor, this transmissible disease is just described for the first time.People and domestic animal can be carried the tick sting of this virus and infect, and occur central nervous system symptom, not only cause human diseases and death, domestic animal more can be caused dead, cause financial loss, cause public health problem.
At present, the gold standard of innerland Viral diagnosis is virus purification, but because its complex operation, large, the consuming time length of technical difficulty, requirement for experiment condition are high, be difficult to apply in real work.Widely used conventional nucleic acid amplification and real-time fluorescence quantitative PCR have played vital role in the context of detection of virus in recent years.But, in view of the reasons such as pathogen capacity is lower in tick worm sample basis, set up a kind of quick, sensitive detection technique, be more conducive to the Viral diagnosis that carrying capacity in tick is lower.
Summary of the invention
For solving the problems of the technologies described above, the object of this invention is to provide one group for detecting primer pair and the nucleotide sequence of innerland virus; The present invention also provides a kind of real-time fluorescence quantitative RT-PCR test kit for detecting innerland virus and detection method thereof.The present invention utilizes TaqMan probe real-time fluorescence PCR technology, sets up a kind of quick, responsive detection method, and the monitoring being applied to innerland virus detects.
The present invention is by the following technical solutions:
Detect a real-time fluorescence quantitative RT-PCR primer pair for innerland virus, the nucleotide sequence of described primer pair is as shown in sequence in sequence table 1 and sequence 2.
The real-time fluorescence quantitative RT-PCR nucleic acid of one group of detection innerland virus, this group nucleic acid comprises primer pair, probe and the amplified production as positive control, the nucleotide sequence of described primer pair is as shown in sequence 2 in sequence in sequence table 1 and sequence table, the nucleotide sequence of described probe is as shown in sequence in sequence table 3, and the described amplified production as positive control includes the nucleotide sequence as shown in sequence in sequence table 4.
Detect a real-time fluorescence quantitative RT-PCR test kit for innerland virus, this test kit mainly comprises this test kit and comprises: 2 × RT-PCR reaction solution, 25 × RT-PCR enzyme mixed solution, upstream primer, downstream primer, probe, negative control and positive control; Specifically composed as follows:
2 × RT-PCR reaction solution (2 × RT-PCRbuffer);
25 × RT-PCR enzyme mixed solution (25 × RT-PCREnzymeMix);
Upstream primer: nucleotide sequence is as shown in sequence in sequence table 1;
Downstream primer: nucleotide sequence is as shown in sequence in sequence table 2;
Probe: nucleotide sequence is as shown in sequence in sequence table 3, and 5 ＇ ends of described probe connect fluorophor, and 3 ＇ ends connect quenching group;
Negative control: nuclease free sterilizing ultrapure water;
Positive control: include the gene of nucleotide sequence as shown in sequence in sequence table 4.
Test kit as above, preferably, described fluorophor be in FAM, CY3 any one, described quenching group be in BHQ1, TAMARA and BHQ2 any one.
Utilize TaqMan probe real-time fluorescent quantitative RT-PCR method to detect a Non-diagnostic method for innerland virus, the method comprises the following steps:
(1) from sample, viral RNA is extracted;
(2) real-time fluorescence RT-PCR amplification is carried out to the RNA extracted, wherein, reaction system is formulated as follows: 1 × RT-PCR reaction solution (1 × RT-PCRBuffer), 1 × RT-PCR enzyme mixed solution (1 × RT-PCREnzymeMix), upstream primer: nucleotide sequence is as shown in sequence in sequence table 1; Downstream primer: nucleotide sequence is as shown in sequence in sequence table 2; Probe: nucleotide sequence is as shown in sequence in sequence table 3, and described probe 5 ＇ holds and connects fluorophor, 3 ＇ ends connect quenching group; Upstream and downstream primer and concentration and probe concentration are 0.01 ~ 1 μM, sample RNA; Arrange nuclease free sterilizing ultrapure water is negative control simultaneously;
Real-time fluorescence RT-PCR response procedures:
(3) collect fluorescent signal, select the fluoroscopic examination pattern of above-mentioned fluorophor, baseline adjustment gets the fluorescent signal of 3 ~ 15 circulations, exceedes the vertex setting threshold line of normal negative control with threshold line;
(4) result judges: testing sample fluorescence growth curve exceedes threshold line, and presents good logarithm growth, be then judged as the positive, as without typical amplification curve, be judged as feminine gender.
Beneficial effect of the present invention:
The present invention establishes the method for quick than more efficient, sensitive innerland virus, provides real-time fluorescent RT-PCR detection reagent box.The detection kit provided is easy to use, easy and simple to handle, and level of automation is high, effectively substitutes traditional virus purification and obtains detected result, and test kit reagent used is few, enormously simplify operating process, and decreases the pollution in operating process, Detection results is good, and high specificity is highly sensitive.Detection kit minimum detectability of the present invention is 14.9 × 10
-10ng/ μ L.
Accompanying drawing explanation
Fig. 1 is the electrophorogram that innerland virus regular-PCR detects.
Fig. 2 is the amplification curve that PCR kit for fluorescence quantitative of the present invention detects innerland virus.
Fig. 3 is innerland FLuorescent quantitative PCR specific amplification curve.
Fig. 4 is the amplification typical curve that PCR kit for fluorescence quantitative of the present invention detects innerland virus.
Fig. 5 utilizes test kit of the present invention to the detected result of sample.
Embodiment
Below in conjunction with specific examples, the present invention is described in further details; not limitation of the invention; embodiments of the present invention are not limited to this; the complementary sequence of nucleotide sequence provided by the invention also can realize the present invention; if no special instructions; agents useful for same is conventional reagent, and therefore all equivalent replacements according to this area done by present disclosure, all belong to protection scope of the present invention.
Embodiment 1: regular-PCR detects innerland virus
In primer of the present invention and probe design, the conserved sequence of screening innerland virus S gene fragment, design a pair oligonucleotide sequences (primer) and probe, utilize primer can to increase specifically the S gene fragment of innerland virus, primer amplification clip size is 113bp, and nucleotide sequence is as shown in sequence in sequence table 4.
Sequence 4:
GTGAGAGCAAATACAATGATGTACTTCACATCATTCCTCCAGTTCTCACCCCCCCTCTCACGAAGCAGCCCAAAGATCACAGCAGGATCCAGGCCCTCATAAGCTATCTCCT。
Primer and the probe of design are as follows:
HRTV-F (in sequence table sequence 1): 5 '-GTGAGAGCAAATACAATGA-3 '
HRTV-R (in sequence table sequence 2): 5 '-AGGAGATAGCTTATGAGG-3 '
HRTV-FAM (in sequence table sequence 3): 5 '-TCACATCATTCCTCCAGTTCTCACC-3 '
The primer pair innerland virus of Application Design carries out regular-PCR amplification, adopts 25 μ L reaction systems: HRTV-F, HRTV-R primer final concentration is respectively 0.4 × 10
3nmol/L; Mg
2+final concentration 2mmol/L; DNTP final concentration is 0.8 × 10
6μm ol/L; 1 × RT-PCR reaction solution (1 × RT-PCRBuffer); Taq polysaccharase consumption 1 unit (unit), template 2 μ L.
Pcr amplification reaction condition is:
95 DEG C of denaturations 3 minutes, then carry out circulating reaction: 95 DEG C of sex change 30 seconds, 49 DEG C of annealing 30 seconds, and 72 DEG C extend 30 seconds, carry out 40 circulations altogether; Last 72 DEG C extend 10 minutes;
Electrophoresis is carried out to amplified production: negative control is sterile saline; Positive control is for carrying the plasmid DNA of sequence as shown in sequence 4, and initial concentration is 14.9 × 10
-3ng/ μ L, and carry out gradient dilution by 10 multiple proportions.Result as shown in Figure 1, can see that designed primer can effectively increase innerland virus.
Embodiment 2: the preparation of innerland virus real-time fluorescence RT-PCR detection kit
Innerland virus real-time fluorescence RT-PCR detection kit comprises following composition:
2 × RT-PCR reaction solution (2 × RT-PCRBuffer);
25 × RT-PCR enzyme mixed solution (25 × RT-PCREnzymeMix);
Upstream primer: nucleotide sequence is as shown in sequence in sequence table 1;
Downstream primer: nucleotide sequence is as shown in sequence in sequence table 2;
Probe: nucleotide sequence is as shown in sequence in sequence table 3;
Negative control: nuclease free sterilizing ultrapure water;
Positive control: the plasmid DNA carrying amplified production, the nucleotide sequence of described amplified production is as shown in sequence in sequence table 4.
Wherein, described 2 × RT-PCR reaction solution and 25 × RT-PCR enzyme mixed solution can select ABI company AgPath-IDTMOne-stepRT-PCRKit; Primer, probe, plasmid entrust the synthesis of Shanghai Ying Jun Bioisystech Co., Ltd, and probe probe 5 ＇ end when synthesizing connects FAM fluorophor and is connected BHQ1 quenching group with 3 ＇ ends.
Embodiment 3: the method that innerland virus real-time fluorescence RT-PCR detects
The present invention detects innerland viral methods and comprises the following steps:
(1) viral RNA extracts
Viral RNA extracts can select QIAampViralRNA extraction test kit to extract.
(2) RT-PCR amplification
The test kit configuration reaction system adopting embodiment 2 to prepare, its component and volume thereof are in table 1.
Table 1 reaction system
Amplification condition: adopt following amplification condition.
(3) collect fluorescent signal, select the fluoroscopic examination pattern of FAM, baseline adjustment gets the fluorescent signal of 3 ~ 15 circulations, with the vertex setting threshold line of threshold line just above normal negative control;
(4) result judges: testing sample fluorescence growth curve exceedes threshold line, and presents good logarithm growth, be then judged as the positive, namely contain innerland viral nucleic acid in sample, as without typical amplification curve, be judged as feminine gender.
In method of the present invention, probe is connected with quenching group with fluorescence, and described fluorescence-quenching group is FAM-BHQ1, and other fluorescent quenching moiety combinations is suitable for too, such as CY3-TAMARA, CY3-BHQ2, FAM-BHQ2, CY3-BHQ1 etc., select corresponding fluoroscopic examination pattern during detection.
Above-mentioned RT-PCR reacts spendable instrument and comprises ABI real-time PCR system (such as 7000,7300,7500,7900 etc.); BioRad Real Time PCR Detection System, Stratagene quantitative polumerase chain reaction instrument (such as MX4000, MX3000, MX3005) etc.
Embodiment 4: the detection of test kit sensitivity of the present invention
Select the innerland virus particle containing extension increasing sequence as positive criteria product, by the DNA of extraction from 10
-1be diluted to 10 successively
-11detect; Innerland virus particle is synthesized by the raw work in Shanghai.
1) positive control: the tick RNA that innerland virus is negative;
2) negative control (NTC): nuclease free water;
3) reaction system is prepared according to table 1 in embodiment 3, and each gradient does three Duplicate Samples.
Detected result as shown in Figure 2.In Fig. 2,1-5 curve is respectively standard substance nuclease free water concentration extent of dilution is from left to right 10
-3-10
-11the curve of amplification.Result judges: Ct value as positive findings, is negative findings lower than 35 more than 38.
Can reach a conclusion from figure, the detection sensitivity of primer and probe in detecting positive criteria product is 14.9 × 10
-10ng/ μ L.
Embodiment 5: the specific detection of test kit of the present invention
1) select innerland virus particle as positive control (PTC);
2) negative control: nuclease free water;
3) template: select heating companion thrombocytopenic syndromes virus, hantaan virus, Seroul virus to carry out non-specific test, above-mentioned template provides by health quarantine institute of China Inst. of Quarantine Inspection Sciences.
4) reaction system is prepared according to table 1 in embodiment 3.
Detected result is as Fig. 3, and result shows, the innerland virus universal real time fluorescent PCR method set up and test kit thereof do not have specific amplification for other viruses except the virus of innerland, show primer and probe design high specificity.
Embodiment 6: the making of innerland virus real-time fluorescence PCR typical curve
1) select innerland virus-positive plasmid as standard substance, by its concentration from 14.9 × 10
-3ng/ μ L is diluted to 14.9 × 10 successively
-11ng/ μ L, selects 10
-3-10
-11as template;
2) negative control: sterilizing ultrapure water;
3) reaction system is prepared according to table 1 in embodiment 3.
As shown in Figure 4, the typical curve of innerland virus universal real time fluorescent PCR is detected result: R
2=0.9959; Wherein, X-axis represents the corresponding logarithm correcting rear reporter fluorescence intensity, Y-axis cycle index.
Embodiment 7: test kit of the present invention detects unknown sample
Utilize test kit of the present invention, detect Black Hills carrapato worm sample these 88 parts, specific experiment step is as follows:
(1) viral RNA extracts
Viral RNA extraction can be selected sky to follow DNA, RNA to extract test kit altogether and be extracted.
A. homogenized:
In tick worm sample basis, add appropriate lysate RLplus, with electronic or glass homogenizer, sample is thoroughly ground, vortex concussion 30sec.
B.12,000rpm (13,400 × g) centrifugal 3-5min, careful supernatant of drawing is to adsorption column CB3 (adsorption column CB3 is placed in 2ml centrifuge tube), and 12,000rpm (13,400 × g) is centrifugal.
Total RNAs extraction
C. in filtrate, 1 times of volume 70% ethanol is added, mixing.
D. the solution obtained proceeds to (adsorption column CR3 puts into collection tube) in adsorption column CR3 together with precipitation, and 12,000rpm (13,400 × g) centrifugal 30-60sec, outwells the waste liquid in collection tube, put back in collection tube by adsorption column CR3.
Attention: when solution and precipitation are transferred to adsorption column CR3, as fruit volume is greater than adsorption column capacity, can make two bites at a cherry.
E. in adsorption column CR3, add 700 μ l protein liquid removal RW1,12,000rpm (13,400 × g) centrifugal 30-60sec, outwells the waste liquid in collection tube, is put back in collection tube by adsorption column CR3.
F. in adsorption column CR3, add 500 μ l rinsing liquid RW (please first check whether before use and added ethanol), room temperature leaves standstill 2min, and 12,000rpm (13,400 × g) centrifugal 30-60sec, outwells the waste liquid in collection tube, is put back in collection tube by adsorption column CR3.
G. repeating step f.
H.12,000rpm (13,400 × g) centrifugal 2min, outwells waste liquid.Adsorption column CR3 is placed in room temperature and places several minutes, thoroughly to dry rinsing liquid remaining in sorbing material.
I. adsorption column CR3 is proceeded in a new 1.5mlRNase-Free centrifuge tube, add 30-100 μ lRNase-FreeddH
2o room temperature places 2min, 12,000rpm (13,400 × g) centrifugal 2min, obtains RNA solution.
(2) real-time fluorescence RT-PCR amplification
The test kit configuration reaction system adopting embodiment 2 to prepare, its component and volume thereof are in table 2.
Table 2 reaction system
Amplification condition: adopt following amplification condition.
(3) collect fluorescent signal, select the fluoroscopic examination pattern of FAM, baseline adjustment gets the fluorescent signal of 3 ~ 15 circulations, with the vertex setting threshold line of threshold line just above normal negative control; The threshold line selected be specially 17.32.
(4) result judges: testing sample fluorescence growth curve exceedes threshold line, and presents good logarithm growth, be then judged as the positive, namely contain innerland viral nucleic acid in sample, as without typical amplification curve, be judged as feminine gender.
In method of the present invention, probe is connected with quenching group with fluorescence, and described fluorescence-quenching group is FAM-BHQ1.
In 35 parts of tick worm samples bases, no positive sample detects, and detected result as shown in Figure 5, proves that test kit detected result of the present invention accurately, reliably.
Can be seen by above specific embodiment, real-time fluorescence quantitative PCR detection kit provided by the invention is easy to use, test kit reagent used is few, enormously simplify operating process, decrease the pollution in operating process, detection specificity is strong, highly sensitive, is applicable to rapid detection innerland virus.
Claims (6)
1. detect a real-time fluorescence quantitative RT-PCR primer pair for innerland virus, it is characterized in that, the nucleotide sequence of described primer pair is as shown in sequence in sequence table 1 and sequence 2.
2. one group is detected the real-time fluorescence quantitative RT-PCR nucleic acid of innerland virus, it is characterized in that, this group nucleic acid comprises primer pair, probe and the amplified production as positive control, the nucleotide sequence of described primer pair is as shown in sequence 2 in sequence in sequence table 1 and sequence table, the nucleotide sequence of described probe is as shown in sequence in sequence table 3, and the described amplified production as positive control includes the nucleotide sequence as shown in sequence in sequence table 4.
3. detect a real-time fluorescence quantitative RT-PCR test kit for innerland virus, it is characterized in that, this test kit comprises: 2 × RT-PCR reaction solution, 25 × RT-PCR enzyme mixed solution, upstream primer, downstream primer, probe, negative control and positive control; The nucleotide sequence of described upstream primer is as shown in sequence in sequence table 1; The nucleotide sequence of described downstream primer is as shown in sequence in sequence table 2; The nucleotide sequence of described probe is as shown in sequence in sequence table 3, and 5 ＇ ends of described probe connect fluorophor, and 3 ＇ ends connect quenching group; Described negative control is nuclease free sterilizing ultrapure water; Described positive control is for including the gene of nucleotide sequence as shown in sequence in sequence table 4.
4. the real-time fluorescence quantitative RT-PCR test kit of detection innerland according to claim 3 virus, is characterized in that, described fluorophor is FAM or CY3, and described quenching group is BHQ1, TAMARA or BHQ2.
5., for detecting a real-time fluorescence quantitative RT-PCR detection method for innerland virus, it is characterized in that, the method comprises the following steps:
(1) from sample, viral RNA is extracted;
(2) carry out real-time fluorescence RT-PCR amplification to the RNA extracted, reaction system is: 1 × RT-PCR reaction solution, 1 × RT-PCR enzyme mixed solution, upstream primer: nucleotide sequence is as shown in sequence in sequence table 1; Downstream primer: nucleotide sequence is as shown in sequence in sequence table 2; Probe: nucleotide sequence is as shown in sequence in sequence table 3, and described probe 5 ＇ holds and connects fluorophor, 3 ＇ ends connect quenching group; Upstream and downstream primer and concentration and probe concentration are 0.01 ~ 1 μM; Simultaneously with nuclease free sterilizing ultrapure water for negative control;
Real-time fluorescence RT-PCR response procedures is:
(3) collect fluorescent signal, select the fluoroscopic examination pattern of fluorophor, baseline adjustment gets the fluorescent signal of 3 ~ 15 circulations, exceedes the vertex setting threshold line of normal negative control with threshold line;
(4) result judges: testing sample fluorescence growth curve exceedes threshold line, and presents good logarithm growth, be then judged as the positive, as without typical amplification curve, be judged as feminine gender.
6. the real-time fluorescence quantitative RT-PCR detection method for detecting innerland virus according to claim 5, is characterized in that, described fluorophor is FAM or CY3, and described quenching group is BHQ1, TAMARA or BHQ2.
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| CN102634604A (en) * | 2012-02-06 | 2012-08-15 | 河南省疾病预防控制中心 | Reagent kit for detecting new Bunyavirus by real-time fluorescent RT-PCR (reverse transcription-polymerase chain reaction) |
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| CN102634604A (en) * | 2012-02-06 | 2012-08-15 | 河南省疾病预防控制中心 | Reagent kit for detecting new Bunyavirus by real-time fluorescent RT-PCR (reverse transcription-polymerase chain reaction) |
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| Title |
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| MATSUNO K ET AL.: "Comprehensive Molecular Detection of Tick-Borne Phleboviruses Leadsto the Retrospective Identification of Taxonomically UnassignedBunyaviruses and the Discovery of a Novel Member of the Genus Phlebovirus", 《JOURNAL OF VIROLOGY》 * |
| SAVAGE HM ET AL.: "First Detection of Heartland Virus (Bunyaviridae: Phlebovirus) from Field Collected Arthropods", 《AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE》 * |
| 于浩等: "《生物信息学实验指导》", 30 September 2009 * |
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