CN102424866A - Fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for rapidly detecting XHF (Xinjiang hemorrhagic fever) viruses - Google Patents
Fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for rapidly detecting XHF (Xinjiang hemorrhagic fever) viruses Download PDFInfo
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Abstract
The invention relates to a fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for rapidly detecting XHF (Xinjiang hemorrhagic fever) viruses. In the invention, a simple, rapid and sensitive RT-PCR detection kit is developed on the basis of designing a specific primer according to a specific conserved sequence of an XHF virus and successfully establishing a PCR detection method for XHF viruses. When XHF viruses are detected by using the kit and detection method provided by the invention, the detection process is convenient and rapid, a detection result can be obtained in 2-3 hours, and the detection sensitivity and the detection accuracy are high, therefore, the kit and detection method provided by the invention are especially suitable for the needs of entry-exit inspection and quarantine.
Description
Technical field
The invention belongs to the molecular Biological Detection field, particularly a kind of fluorescence quantitative RT-PCR kit of rapid detection xinjiang hemorrhagic fever virus, the detection method of test kit and this test kit are in application clinical, the Check and Examination of Port xinjiang hemorrhagic fever virus.
Background technology
Xinjiang hemorrhagic fever (Xinjian hemorrhagic fever; XHF) be xinjiang hemorrhagic fever virus (Xinjian hemorrhagic fever virus by the bunyaviridae Nairovirus; Be called for short XHFV, claim crimean-Congo hemorrhagic fever virus again) acute infectious disease that causes, propagate through the tick matchmaker; Mainly be popular in three continents, Europe, Asia and Africa; Xinjiang hemorrhagic fever obvious seasonal arranged, be epidemic peak annual 4～May, is consistent busy season in natural growth and decline situation and pastoral area active with tick.Clinical symptoms has heating, headache, sleepy weak, vomiting, blood urine, proteinuria even shock etc.At present Xinjiang hemorrhagic fever is not still had specific treatment, principle should be taked complex treatment measure, is main with control over bleeding and shock.Therefore early stage quick diagnosis is a disease controlling, improves curative ratio, avoids clinical misdiagnosis to affect the treatment key on opportunity adversely.
Conditions such as the geography of China some areas, weather, communication media-tick, contagium are similar with popular district, three continents, Europe, Asia and Africa; Along with development of global economy; International crowd contacts are more convenient, and trend such as the integrated and life that urbanizes of global trade make that virus and communication media more are prone between each country, propagate.So be necessary to set up the rapid molecular biological detection method of xinjiang hemorrhagic fever virus; In the frontier port entry and exit crowd and media biology are detected fast in early days and monitor; Prevent this transmissible disease importing at the port; Accomplish the propagation of timely prevention, control disease, Economic development and the people's health that ensures China had great significance.
Domestic research to xinjiang hemorrhagic fever virus at present is less, only set up should virus tissue culture and RT-PCR detection method.The molecular biology method that the diagnostic method of xinjiang hemorrhagic fever virus is mainly comprised tissue culture, serodiagnosis and new development abroad.The cultivation of xinjiang hemorrhagic fever virus requires breadboard Biosafety grade high, needs to carry out in the BSL-3 laboratory, and the time that virus is separated, identified is longer, and detection sensitivity is not high; Serological method mainly comprises (1) complement fixation test (CFT): mainly detect complement fixation antibody, it is slower that positive reaction occurs, the less employing of clinical labororatory; (2) neutralization test: method is very complicated, generally shall not be applied to clinical; (3) the paired sera sample need is gathered in hemagglutination-inhibition test, is with the antibody titer of second part of serum that to raise more than 4 times be the recent infection index.In addition, still there is report to adopt and catches method detection specificity antibody such as ELISA, indirect immunofluorescence.
Advantages such as molecular biology method is compared with tissue culture, serodiagnosis, has the detection sensitivity height, and high specificity and detection time are short, but the fluorescence quantitative RT-PCR kit of still suitable at present port rapid detection xinjiang hemorrhagic fever virus.
Summary of the invention
The objective of the invention is to overcome above-mentioned technical disadvantages, a kind of fluorescence quantitative RT-PCR kit and detection method of rapid detection xinjiang hemorrhagic fever virus is provided, the quick test of an xinjiang hemorrhagic fever virus such as, port clinical to be fit to.
The fluorescence quantitative RT-PCR kit of detection xinjiang hemorrhagic fever virus of the present invention, it comprises:
(1) test kit selected for use of RT-PCR reaction is AgPath-ID One-Step RT-PCR Kit (U.S. Ambion Company products).Include 25 * RT-PCR enzyme mixed solution: comprise counter-rotating enzyme, warm start Taq enzyme; 2 * RT-PCR reaction the buffer and the ROX positive are with reference to dyestuff.
(2) forward primer 100 μ L, concentration is 12.5 μ M;
(3) reverse primer 100 μ L, concentration is 17.5 μ M;
(4) fluorescent probe 50 μ L, concentration is 12.5 μ M;
(5) aseptic DEPC water: 5mL
(6) positive control: promptly contain the special gene RNA segment of xinjiang hemorrhagic fever virus;
(7) negative control: aseptic DEPC treating water.
Said primer is the synthetic dna fragmentation to reaching fluorescent probe, and base sequence is respectively:
Forward primer: 5 '-TGACAGCATT TCTTTAACAGACATCA-3 ',
Reverse primer: 5 '-AAACACGGCAGCCTTAAGCA-3 ',
Fluorescent probe: 5 '-FAM-TCGCCAGGGACTTTATATTCTGCAAGG-TAMRA-3 '.
Above-mentioned primer, probe are the xinjiang hemorrhagic fever virus nucleotide sequences that obtains from Genbank with the SeqMan programanalysis in the Lasergene software package; Selection has the special zone of Nucleotide; The Auele Specific Primer and the probe that obtain with Primer Express2.0 software design; This primer and the probe basis on location on the xinjiang hemorrhagic fever virus genome is that the GenBank sequence number is the strain sequence of HQ833035.1, and relevant information sees table 1 for details.
Table 1
Use the method that the mentioned reagent box detects xinjiang hemorrhagic fever virus, carry out as follows:
(1) RNA of extraction testing sample;
(2) RT-PCR amplification, method is following:
In RT-PCR reagent pipe, add 25 * RT-PCR enzyme mixed solution, 1 μ l, 2 * RT-PCR reaction buffer12.5 μ l, forward primer 1 μ l; Reverse primer 1 μ l; Fluorescent probe 0.5 μ l, template ribonucleic acid 100pg to 1 μ g (≤5 μ l) is supplemented to 25 μ l with the DEPC treating water; Instantaneous centrifugal mixing; Set up negative control and positive control to put the amplification of RT-PCR appearance together simultaneously; Amplification condition is: 45 ℃ 10 minutes, 95 ℃ after 15 minutes, get into amplification cycles, 95 ℃ 15 seconds, 60 ℃ of circulations in 45 seconds 40 times;
(3) RT-PCR result judges:
No Ct value or Ct＞45, and do not have obvious amplification curve, be judged to be feminine gender;
Ct≤40, and obvious amplification curve is arranged, be judged to be the positive;
40＜Ct≤45 are detected again, and are then positive if see that obvious amplification curve is arranged, otherwise negative.
Testing sample can have two types: (1) human serum sample, directly extract and carry out the fluorescence RT-PCR operation behind the viral RNA; (2) communication media sample-tick carries out the fluorescence RT-PCR operation after extracting viral RNA after usually grinding again, relates to the pre-treatment that tick grinds; Use Hank ' s liquid as lapping liquid; Grind testing sample, the fragment of tissue disappearance to testing sample obtains ground and mixed liquid.
The present invention has following advantage:
The present invention is at design xinjiang hemorrhagic fever virus special primer and successfully set up on the basis of PCR detection method of xinjiang hemorrhagic fever virus, is developed into the RT-PCR detection kit of simple fast sensitive.Compare with other xinjiang hemorrhagic fever virus detection technique; Detect xinjiang hemorrhagic fever virus with test kit of the present invention and detection method following advantage is arranged: (1) testing process is fast convenient; Can learn detected result (other method such as viral stripping technique need 24 hours or more than week at least) in 2-3 hour, the needs of the check inspection and quarantine that is particularly suitable for entering and leaving the border; (2) detection sensitivity is higher, detects lower limit and is low to moderate 2 copies; (3) accuracy that detects is high, and the present invention has guaranteed the accuracy and the specificity that detect according to the peculiar one section conserved sequence design special primer of xinjiang hemorrhagic fever virus and the comparison of increasing.
Description of drawings
Fig. 1 is an experimental result picture of confirming best primer concentration;
Fig. 2 is an experimental result picture of confirming best concentration and probe concentration;
Fig. 3 is the experimental result picture of canonical plotting;
Fig. 4 is the experimental result picture of the repeatability of test kit of the present invention;
Fig. 5 is the product electrophorogram of xinjiang hemorrhagic fever virus RNA moulding plate series dilution regular-PCR amplification;
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.
Embodiment 1: the preparation of positive control RNA template
According to xinjiang hemorrhagic fever virus (GenBank sequence number: sequence HQ833035.1); The size that amplification comprises fluorescence RT-PCR detection fragment sequence is the dna fragmentation of 606bp; Clone on the PMD-18T carrier; Extract the test kit plasmid DNA purification with DNA, order-checking confirms to insert segmental direction.The xinjiang hemorrhagic fever virus sequence of use and carrier paired versatility primer amplification T7 promotor and insertion is carried out in-vitro transcription with MAXIscript T7 Kit subsequently, generates purpose RNA; Carrying out DNase with TURBO DNase then handles; Remove the pcr amplification template of not transcribed, use ethanol sedimentation at last, the purifying RNA product; Be the positive control RNA template, it is for use to be stored in-80 ° of C Ultralow Temperature Freezers.
Get 1 μ L in-vitro transcription template ribonucleic acid, dilute 1000 times, use DU730 to record RNA template original liquid concentration and be 931ng/ μ l.
Again according to the calculation formula " copy number (copies/mL)=RNA concentration (g/mL) * 6.02 * 10 of the copy number of single stranded RNA
23(copies/moL)/(340 * single stranded RNA base number (g/moL)) " calculate: the copy number of in-vitro transcription template ribonucleic acid stoste is about 2.70 * 10
12(copies/ μ L).
Embodiment 2: the confirming of amplification program
The test kit that the RT-PCR reaction is selected for use is AgPath-ID One-Step RT-PCR Kit (U.S. Ambion Company products), and amplification and detection are carried out on ABI7500 real-time fluorescence quantitative PCR instrument.
Concrete property according to primer, probe; Response procedures on the instrument is selected to have tested 3 kinds of temperature and 2 kinds of times in annealing extension condition: 45 ℃ 10 minutes; 95 ℃ after 15 minutes, get into 40 PCR circulations: 95 ℃ of 15 seconds → (58/60/62) ℃ (30/45) second; The application of sample amount of all ingredients is respectively to contain in each reaction tubes and has 25 * RT-PCR enzyme mixed solution, 1 μ l; 2 * RT-PCR reaction buffer12.5 μ l, forward primer 1 μ l, reverse primer 1 μ l; Fluorescent probe 0.5 μ l; The template ribonucleic acid amount is 100pg to 1 μ g (≤10 μ l), supplies TV 25 μ l with the DEPC treating water, and the fluorescent signal collection is located at the annealing extension stage.The all visible positive amplification curve of result, but consider and make primer amplification that better specificity arranged and shorten the reaction times, confirm that at last amplification program is: 45 ℃ 10 minutes, 95 ℃ 15 minutes; 95 ℃ 15 seconds → 60 ℃ 45 seconds, 40 times the circulation.
Embodiment 3: the confirming of best primer concentration
When the probe final concentration was 250nM, by the test of increasing respectively of the listed eight groups of forward and reverse primer concentrations of table 2, test-results made amplification curve such as Fig. 1.It is minimum from Fig. 1, to select the Ct value, and the amplification curve comparatively significantly pairing primer concentration of curve (the 3rd curve among Fig. 1) is an optimum concn, i.e. forward primer final concentration 500nM, and the reverse primer final concentration is 750nM.
Table 2
| Divide into groups | XHFV-FP/RP (final concentration nM) | The |
| 1 | 750/500 | 13.25 |
| 2 | 750/250 | 13.57 |
| 3 | 500/750 | 13.18 |
| 4 | 500/500 | 13.34 |
| 5 | 500/250 | 13.71 |
| 6 | 250/750 | 13.66 |
| 7 | 250/500 | 13.27 |
| 8 | 250/250 | 13.65 |
Embodiment 4: the confirming of best concentration and probe concentration
Use above amplification program; According to the final concentration 500nM of above-mentioned definite best forward primer and the final concentration 750nM of optimal reverse primer; Concentration and probe concentration is chosen as respectively: 62.5nM, 125nM, 250nM, 500nM, 750nM, the test of increasing, test-results such as Fig. 2.Select with Ct value minimum from Fig. 2 result, the amplification curve also the most tangible pairing concentration and probe concentration of curve is an optimum concn.See Fig. 2; The Ct value of concentration and probe concentration fluorescence RT-PCR from low to high is respectively 14.45,13.29,12.82,12.36 and 12.80; Various Ct value basically identicals; The highest with 750nM final concentration fluorescence intensity, final concentration 250nM also has higher fluorescence intensity simultaneously, finally confirms xinjiang hemorrhagic fever virus probe final concentration 250nM (the 3rd curve among Fig. 2).
Through above-mentioned experiment, finally confirmed to adopt this test kit to detect this viral concrete grammar:
(1) RNA of extraction testing sample;
(2) RT-PCR amplification, method is following:
In RT-PCR reagent pipe, add 25 * RT-PCR enzyme mixed solution, 1 μ l, 2 * RT-PCR reaction buffer12.5 μ l, forward primer 1 μ l; Reverse primer 1 μ l; Fluorescent probe 0.5 μ l, template ribonucleic acid 100pg to 1 μ g (≤5 μ l) is supplemented to 25 μ l with the DEPC treating water; Instantaneous centrifugal mixing; Set up negative control and positive control to put the amplification of RT-PCR appearance together simultaneously; Amplification condition is: 45 ℃ 10 minutes, 95 ℃ after 15 minutes, get into amplification cycles, 95 ℃ 15 seconds, 60 ℃ of circulations in 45 seconds 40 times;
(3) RT-PCR result judges:
No Ct value or Ct＞45, and do not have obvious amplification curve, be judged to be feminine gender;
Ct≤40, and obvious amplification curve is arranged, be judged to be the positive;
40＜Ct≤45 are detected again, and are then positive if see that obvious amplification curve is arranged, otherwise negative.
Embodiment 5: sensitivity, detectability and typical curve
The xinjiang hemorrhagic fever virus RNA positive template of in-vitro transcription carries out 10 times of serial dilutions (2.70 * 10 with the DEPC treating water
11, 2.70 * 10
10, 2.70 * 10
9, 2.70 * 10
8, 2.70 * 10
7, 2.70 * 10
6, 2.70 * 10
5, 2.70 * 10
4, 2.70 * 10
3, 2.70 * 10
2, 2.70 * 10
1, 2.70 * 10
0Copies/ μ l), detect through optimizing good reaction system, the result finds 2.70 * 10
1Copies/ μ l and 2.70 * 10
0Though the RNA template of copies/ μ l has tangible amplification curve, the Ct value is greater than 40, promptly minimumly detects 2.70 * 10
0The dilution RNA of copies/ μ l, LDL are about 2 copies/ reaction, and linearity range has 10 one magnitude (2.70 * 10
2～2.70 * 10
11Copies/ μ l); Detected result according to these 10 extent of dilution template ribonucleic acids generates typical curve automatically by software, and getting typical curve Y=-3.308X+57.59 slope is-3.308, sees Fig. 3; Fig. 3 is the typical curve that the xinjiang hemorrhagic fever virus fluorescence RT-PCR detects, according to " E=10
-1/ slope, % efficient=(E-1) * 100% " calculate, pcr amplification efficient is 99.5%.
Embodiment 6: reperformance test
In-vitro transcription xinjiang hemorrhagic fever virus RNA template is diluted with water to 2.70 * 10
10, 2.70 * 10
7, 2.70 * 10
4The high, medium and low three kinds of concentration of copies/ μ L, every kind of concentration duplicate detection 3 times is carried out with optimizing good reaction system; The result sees Fig. 4, and visible by Fig. 4, three groups of experimental results all have good repeatability; Amplification curve Ct value difference is different very little in the group; Its slope and R2 see table 3, and table 3 shows that for the repeatability of real-time fluorescence RT-PCR method detection xinjiang hemorrhagic fever virus, data amplification curve Ct value difference is different very little in the group.
Table 3
Embodiment 7: sensitivity and specificity test
Detect and arbovirusess such as the similar yellow fever virus in xinjiang hemorrhagic fever virus route of transmission, japanese encephalitis virus with the fluorescence RT-PCR method, the result is all negative, explains that present method has specificity preferably.
For studying fluorescence quantitative PCR method and conventional PCR sensitivity at the check xinjiang hemorrhagic fever virus, utilize identical template, promptly in-vitro transcription xinjiang hemorrhagic fever virus RNA positive template water carries out 10 times of serial dilutions (2.70 * 10
11, 2.70 * 10
10, 2.70 * 10
9, 2.70 * 10
8, 2.70 * 10
7, 2.70 * 10
6, 2.70 * 10
5, 2.70 * 10
4, 2.70 * 10
3, 2.70 * 10
2, 2.70 * 10
1, 2.70 * 10
0Copies/ μ l), reverse transcription becomes cDNA, uses TaKaRa Taq
TMReagent (TaKaRa Biotechnology company provides) carries out conventional PCR, and is the same with the fluorescence RT-PCR reaction system, forward primer 1ul; Reverse primer 1.5 μ L, template cDNA adds 1 μ L equally, reaction TV 20 μ L; All the other reagent are pressed the test kit description operation; Reaction conditions is following: 94 ℃ of preparatory sex change are after 3 minutes, 94 ℃ 1 minute → 60 ℃ 1 minute → 72 ℃ 1 minute 30 times circulations, and 72 ℃ were extended 10 minutes.Amplified production utilizes the Alpha gel imaging system to take a picture through 1.5% agarose, 110 volts of electrophoresis in 1 * TAE after 20 minutes, and the result sees Fig. 5, and Fig. 5 is xinjiang hemorrhagic fever virus RNA moulding plate series dilution RT-PCR amplified production electrophorogram.Among Fig. 5, sign 1,2,3,4,5,6,7,8,9,10,11, the 12 corresponding template cDNA concentration (the copies/ μ l of unit) of expression respectively is: 2.70 * 10
11, 2.70 * 10
10, 2.70 * 10
9, 2.70 * 10
8, 2.70 * 10
7, 2.70 * 10
6, 2.70 * 10
5, 2.70 * 10
4, 2.70 * 10
3, 2.70 * 10
2, 2.70 * 10
1, 2.70 * 10
0M represents 1000bp Marker.Visible from Fig. 5, when cDNA concentration is 2.70 * 10
0During copies/ μ L, the band of purpose amplified fragments is very fuzzy, can not reach effective judgement, and its sensitivity is approximately than low 10 times of fluorescence RT-PCR method.
Claims (2)
1. the fluorescence quantitative RT-PCR kit of rapid detection xinjiang hemorrhagic fever virus is characterized in that comprising following reagent:
(1) RT-PCR reaction kit includes 25 * RT-PCR enzyme mixed solution: comprise counter-rotating enzyme, warm start Taq enzyme; 2 * RT-PCR reaction the buffer and the ROX positive are with reference to dyestuff;
(2) forward primer 100 μ L, concentration is 12.5 μ M;
(3) reverse primer 100 μ L, concentration is 17.5 μ M;
(4) fluorescent probe 50 μ L, concentration is 12.5 μ M;
(5) aseptic DEPC water: 5mL;
(6) positive control: the plasmid that promptly contains the special gene segment of xinjiang hemorrhagic fever virus;
(7) negative control: aseptic DEPC treating water;
Said primer is the synthetic dna fragmentation to reaching fluorescent probe, and base sequence is respectively:
Forward primer: 5 '-TGACAGCATT TCTTTAACAGACATCA-3 ',
Reverse primer: 5 '-AAACACGGCAGCCTTAAGCA-3 ',
Fluorescent probe: 5 '-FAM-TCGCCAGGGACTTTATATTCTGCAAGG-TAMRA-3 '.
2. adopt the said test kit of claim 1 to detect the method for xinjiang hemorrhagic fever virus:
(1) RNA of extraction testing sample;
(2) RT-PCR amplification, method is following:
In RT-PCR reagent pipe, add 25 * RT-PCR enzyme mixed solution, 1 μ l, 2 * RT-PCR reaction buffer12.5 μ l, forward primer 1 μ l; Reverse primer 1 μ l; Fluorescent probe 0.5 μ l, template ribonucleic acid 100pg to 1 μ g (≤5 μ l) is supplemented to 25 μ l with the DEPC treating water; Instantaneous centrifugal mixing; Set up negative control and positive control to put the amplification of RT-PCR appearance together simultaneously: amplification condition is: 45 ℃ were reacted 10 minutes; 95 ℃ were reacted 15 minutes; Get into amplification cycles, 95 ℃ were reacted 15 seconds, and 60 ℃ were reacted 45 seconds, and circulated 40 times;
(3) RT-PCR result judges:
No Ct value or Ct＞45, and do not have obvious amplification curve, be judged to be feminine gender;
Ct≤40, and obvious amplification curve is arranged, be judged to be the positive;
40＜Ct≤45 are detected again, and are then positive if see that obvious amplification curve is arranged, otherwise negative.
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|---|---|---|---|---|
| CN109852730A (en) * | 2019-03-26 | 2019-06-07 | 中国科学院合肥物质科学研究院 | Kit and detection method a kind of while that detect potent virus and bacterium |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191338A (en) * | 2010-12-24 | 2011-09-21 | 中国检验检疫科学研究院 | Fluorescence quantitative polymerase chain reaction (PCR) new method for detecting multiple viruses of yellow fever, dengue fever and epidemicencephalitis B and multiple virus detection PCR system |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191338A (en) * | 2010-12-24 | 2011-09-21 | 中国检验检疫科学研究院 | Fluorescence quantitative polymerase chain reaction (PCR) new method for detecting multiple viruses of yellow fever, dengue fever and epidemicencephalitis B and multiple virus detection PCR system |
Non-Patent Citations (1)
| Title |
|---|
| ANNA PAPA等: "Viral Load and Crimean-Congo Hemorrhagic Fever", 《EMERGING INFECTIOUS DISEASES》, vol. 13, no. 5, 31 May 2007 (2007-05-31) * |
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|---|---|---|---|---|
| CN109852730A (en) * | 2019-03-26 | 2019-06-07 | 中国科学院合肥物质科学研究院 | Kit and detection method a kind of while that detect potent virus and bacterium |
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Application publication date: 20120425 |