CN102140528A - New fluorescence quantitative polymerase chain reaction (PCR) detection method for rift valley fever virus and rift valley fever virus detection PCR system - Google Patents
New fluorescence quantitative polymerase chain reaction (PCR) detection method for rift valley fever virus and rift valley fever virus detection PCR system Download PDFInfo
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Abstract
The invention discloses a new fluorescence quantitative polymerase chain reaction (PCR) detection method for rift valley fever virus and a rift valley fever virus detection PCR system. The system consists of primers and a probe, Premix Ex Taq reaction solution, and sterile Tris water, wherein the primers and the probe have good specificity and high sensitivity, and are suitable for no cross reaction of the rift valley fever and Dengue virus.
Description
Technical field
The invention belongs to field of biological detection, be specifically related to fast qualitative and the quantitative detecting method and the Rift Valley fever virus detection PCR system of Rift Valley fever virus, a pair of Auele Specific Primer and probe are provided.
Background technology
Rift Valley fever (Rift Valley fever, RVF) have another name called the local epidemic hepatitis of sheep and ox infectivity, it is a kind of fatal transmissible disease of African popular, infect the mankind and livestock by mosquito bite, cardinal symptom is acute diarrhea and high fever, and then the liver of grievous injury people and livestock, kidney, part patient also can be dead because of angiorrhexis.International Office of Epizootics (OIE) classifies it as category-A eqpidemic disease.This disease is comparatively serious to the harm of sheep, goat, ox, can cause that pregnant animal miscarriage and new livestock are dead at high proportion, and above the average age for marriage non-pregnant animal is also than susceptible, but clinical disease resistance is stronger.Up to 2000, RVF only was limited to African popular circulation, but had broken out recently the RVF of lethality in the arabia, showed that remarkably urgent need wants quick diagnosis, effectively handles and prevent this disease.The RVF cause of disease is Rift Valley fever virus, and (Rift Valley fever virus RVFV), belongs to Bunyaviridae (Bunyaviridae) Phlebovirus (Phlebovirus).
The detection method that is used for Rift Valley fever virus has at present had virus separations, agarose immunodiffusion(ID), immunofluorescence dyeing, ELISA method, virus neutralization tests, hemagglutination-inhibition test, enzyme linked immunosorbent assay or the like a variety of detection methods, but the method for quantitative fluorescent PCR detection of nucleic acids still is the detection method of generally acknowledged a kind of more directly perceived, a kind of suitable early diagnosis faster.
Summary of the invention
The object of the present invention is to provide a kind of Rift Valley fever virus fluorescence quantitative PCR detection novel method and Rift Valley fever virus to detect the PCR system, this method can fast qualitative and detection by quantitative Rift Valley fever virus, comprises that a pair of specificity is higher, highly sensitive, the primer of good reproducibility and probe.
For achieving the above object, the primer probe that design is adopted in the Rift Valley fever virus fluorescence quantitative PCR detection novel method of the present invention is:
| Name | Sequence | Position | Tm℃ | Modification |
| FP | CATGGATTCTATATTATCAAAACAGC | 18-43 | 57.3 | ? |
| RP | AATGTTGGAAGTGCCAGAGTTAG | 100-122 | 58.2 | ? |
| ?Probe | TGGTTTTGTTAGAGTGCCAATCAAGCA | 57-83 | 68.2 | HEX/BHQ-1 |
Further, described Rift Valley fever virus fluorescence quantitative PCR detection novel method is specially:
1) design primer probe;
2) select fluorescein according to instrument;
3) synthetic primer and probe;
4) preparation positive plasmid standard substance;
5) operation PCR;
6) data analysis;
7) extract viral RNA and carry out the system checking.
Further, described step 2) the selective fluorescent emission group of fluorescein has 6-FAM, TET, HEX, JOE, CY3, CY5, TAMRA, Texas Red in, and the fluorescent quenching group has BHQ-1, BHQ-2, Lowa BlackTMRQ, Lowa BlackTMFQ, Dabcyl.
Further, take the gene synthesis mode to make positive plasmid CBG234-3 as positive in the described step 4): to carry out gene synthetic with prolonging 10～50bp before and after the high conserved region territory bp18-bp122 that filters out in the design of primers process respectively, be cloned into then in the pMD19-T carrier, be described positive plasmid sample, numbering CBG234-3.
Further, the quantitative fluorescent PCR reaction is applicable to all quantitative fluorescent PCR reaction instrument in the described step 5).
Further, described quantitative real time PCR Instrument comprises SmartCyclerII, ABI real-time PCR system, BioRad PCR in real time detection system, Stratagene quantitative polumerase chain reaction instrument.
Further, best amplification condition is in the described step 5):
A kind of detection Rift Valley fever virus PCR system, this system comprise that primer or other can combine the primer that is equal to of also amplification with Rift Valley fever full length sequence specificity, and its similar homology can not surpass 90%, and primer sequence is as follows:
A kind of detection Rift Valley fever virus PCR system, comprise primer and can with Rift Valley fever full length sequence specific probe, probe sequence is as follows:
The a pair of new primer probe of design of the present invention is set up a kind of new quantitative fluorescent PCR system in conjunction with utilization SmartCyclerII and is detected Rift Valley fever virus.Pick out the conservative sequence of Rift Valley fever virus genome camber by the mode of sequence alignment, the a pair of primer of design and a Taqman probe on this sequence, set up the real-time fluorescence quantitative PCR reaction system, and be optimized, detect the sensitivity and the specificity of this method.
Description of drawings
Fig. 1 is that the two dimension of amplification curve shows figure;
Fig. 2 is the screening synoptic diagram of primer concentration and probe concentration;
Fig. 3 detects synoptic diagram for sensitivity;
Fig. 4 is that repeatability detects synoptic diagram;
Fig. 5 is a canonical plotting.
Embodiment
The principle of work of Rift Valley fever virus fluorescence quantitative PCR detection novel method of the present invention promptly is to utilize the variation qualitative analysis of fluorescent signal, detect the variation of each cyclic amplification product amount in the pcr amplification reaction in real time, starting template is carried out quantitative analysis by the relation of Ct value and typical curve.The invention still further relates to all the elements that above-mentioned detection architecture comprises.
This method may further comprise the steps:
1) design primer probe;
2) select fluorescein according to instrument;
3) synthetic primer and probe;
4) preparation positive plasmid standard substance;
5) operation PCR;
6) data analysis;
7) extract viral RNA and carry out the system checking.
1, the design of primer probe
According to intending research gene title Rift Valley fever virus (Rift Valley fever virus, RVFV), in genebank, find corresponding complete genome sequence (www.ncbi.nl m.nih.gov), utilization DNASTAR software carries out homology analysis and blast sequential analysis, sift out high conservative zone bp18-bp122 as target sequence, adopt primer-design software Primer Premier 5 to carry out the design of primer probe, sequence sees Table 1.
The primer and the probe of table 1 fluorescence quantitative PCR detection Rift Valley fever virus
| Name | Sequence | Position | Tm℃ | Modification |
| FP | CATGGATTCTATATTATCAAAACAGC | 18-43 | 57.3 | ? |
| RP | AATGTTGGAAGTGCCAGAGTTAG | 100-122 | 58.2 | ? |
| Probe | TGGTTTTGTTAGAGTGCCAATCAAGCA | 57-83 | 68.2 | HEX/BHQ-1 |
2, the selection of fluorescein
Use the SmartCyclerII of instrument as U.S. Cepheid company, the fluorescent emission group adopts the most stable HEX group of characteristic, and the fluorescent quenching group adopts BHQ-1.
3, the synthetic of primer and probe gives precious biotechnology (Dalian) company limited to finish.
4, the present invention takes the gene synthesis mode to make positive plasmid as positive.To carry out gene synthetic with prolonging 30～50bp before and after the high conserved region territory bp18-bp122 that filters out in the design of primers process respectively, is cloned into then in the pMD19-T carrier, is our positive plasmid sample, numbering CBG234-3.
5, the preparation of plasmid standard
Synthetic by authorized company and extract positive plasmid sample stoste, recording concentration by the ultramicron ultraviolet spectrophotometer is 310ng/ μ l, calculates according to the copy number calculation formula " copy number concentration (copies/ μ l)=DNA concentration (ng/ μ l) * 6.02 * 1023 (copies/mol)/324 * 2 * (carrier base number+goal gene base number) " of single stranded DNA and learns that the DNA copy number concentration of plasmid stoste is about 1 * 10
11Copies/ μ l.Use EASY Dilution (a kind of liquid that is specifically designed to standard substance dilution use, precious biotech firm produces by Dalian) to carry out gradient dilution and finally obtain 1 * 10
0～1 * 10
10The plasmid standard of copies/ μ l.
6, primer and probe dissolved dilution are to desired concn and keep in Dark Place.
Primer generally is diluted to 10 μ m/L, and probe dilution is to 5 μ m/L, and-20 ℃ of refrigerators seal and keep in Dark Place.Dilution institute water is sterilization Tris water (Ph7.0～8.0).
7, Real-Time pcr amplification
The amplification kit of selecting for use is a Premix Ex Taq test kit, available from precious biotechnology (Dalian) company limited.Reaction system component and volume thereof see Table 2.
Table 2
Amplification condition is as follows
8, data analysis
9, extract viral RNA, carry out reverse transcription, gained cDNA uses the aqua sterilisa gradient dilution, replaces positive and carries out detection validation.
The selection of threshold value
In the process of the various parameter optimizations of quantitative fluorescent PCR, the Ct value is the reference factor of most critical.The method of calculating the Ct value has in conjunction with two kinds of method of intersection and quafric curve Peak Intensity Method.We will react the threshold line that presents in the amplification curve diagram and drag up and down, para-curve in conjunction with the amplification rate velocity of variation is observed, when the pairing Ct value of the intersection point of threshold line and amplification curve is passed through the peak value of quafric curve just, the threshold value of this moment is optimal threshold, and the Ct value also is Ct value the most accurately.With reference to figure 1, determine that the body series optimal threshold is 30.The optimization of primer concentration and probe concentration
1. it is standby to lower concentration at first to dilute the primer probe, and primer is diluted to 10 μ m/L, and probe is to 5 μ m/L;
Respectively to each system add 0.25 μ L, 0.5 μ L, 1 μ L has diluted upstream and downstream primer (10 μ m/L), 0.25 μ L, 0.5 μ L, 1 μ L Taqman probe (5 μ m/L) move PCR simultaneously, other components are pressed table 2 preparation in the system; (considering that the system composition too much easily produces and suppress, is the upper limit so the add-on of primer probe is controlled at 1 μ L.)
3. the preferred optimum response concentration combination of relative method, result be as shown in Figure 2: A1, A2, A3 are the amplification curves that system adds 1 μ L primer probe, and fluorescent value shows and surpass 1000 that the Ct value is less; A4, A5, A6 are the amplification curves that adds 0.5 μ L primer probe, and fluorescent value is about about 550, and the Ct value is less; A7, A8, A9 are the amplification curves that adds 0.25 μ L primer probe, and fluorescent value is about in the of 170, and the Ct value is bigger than normal; The negative contrast of A10.Strong according to fluorescent signal, the principle that the Ct value is little seems for first group and be optimum concn, but first group and second group of corresponding threshold are all very high, common empirical value is got 30 and is got final product, and too high threshold value can influence the accuracy of detected result, and the 3rd group of concentration is very suitable for 30 threshold value, and corresponding Ct point is just on the peak point of two-dimensional curve, so in the 25 μ L systems, determine that finally best primer (10 μ m/L) probe (5 μ m/L) concentration adds 0.25 μ L, i.e. 100 * 50nm/L for each.
The selection of annealing temperature
Under the perfect condition, annealing temperature is enough low, effectively anneals with aim sequence to guarantee primer, wants enough high simultaneously, to reduce non-specific binding.Following carrying out when annealing temperature is set: be lower than that design estimates during primer Tm5 ℃ as initial annealing temperature, with 2 ℃ be increment, progressively improve annealing temperature.5 groups of temperature of initial setting: 52 ℃, 54 ℃, 56 ℃, 58 ℃, 60 ℃.All the purpose fragment be can detect under five groups of annealing temperatures, this primer and probe better performances proved, adaptable wider range.But the minimum temperature of the strongest while Ct of fluorescent signal value is 56 ℃, determines that finally optimum annealing temperature is 56 ℃.
The mensuration of sensitivity
Detect gradient template, Fig. 3 according to reaction system of having optimized and response procedures. for middle A1-A6 is 1e
1Copies/ μ l template amplification curve, five positive feminine genders; A7-A15 is 1e
0Copies/ μ l template amplification curve, eight positive feminine genders think that lowest detectable limit can reach number level, 1e
0The dilution DNA of copies/ μ l.
Repeatability detects
Fig. 4 is 1e
6～1e
2The dna profiling quantitative fluorescent PCR of five concentration gradients of copies/ μ l, each gradient have all been done 2 to 3 parallel laboratory tests, and the amplification curve that each gradient template produced among the figure has all produced and has been consistent Ct value, and repeatability is very good.
The specificity check
With the cause of disease dengue virus of the approaching singapore hemorrhagic fever of method detection of being set up and Rift Valley fever clinical manifestation, the result is negative.Show that this designed primer probe specificity is very high.
The foundation of typical curve
The quality of system can reflect that the e value levels off to 1 more by amplification efficiency e, amplification efficiency is described more near 100%, and reaction system is good more; E＜1 illustrates that the system amplification efficiency is lower, and system needs to optimize; Inhibition is contained in the explanation system in e＞1, needs to reduce even get rid of the interference of inhibition.The amplification efficiency optimum range of generally acknowledging in this field is between 0.8-1.2.Adopt 1e
8～1e
4Copies/ μ l template increases, and the gained typical curve is Fig. 5, the amplification function
Y=-0.297X+13.102, amplification efficiency e=10-k-1=0.98=98%.(k＝-0.297)
Purpose of the present invention is exactly a pair of new primer probe of design, sets up a kind of new quantitative fluorescent PCR system in conjunction with utilization SmartCyclerII and detects Rift Valley fever virus.Pick out the conservative sequence of Rift Valley fever virus genome camber by the mode of sequence alignment, the a pair of primer of design and a Taqman probe on this sequence, set up the real-time fluorescence quantitative PCR reaction system, and be optimized, detect the sensitivity and the specificity of this method.Detection method of the present invention is highly suitable for Rift Valley fever virus, with the dengue fever virus no cross reaction.
Claims (9)
1. Rift Valley fever virus fluorescence quantitative PCR detection novel method is characterized in that, the primer probe that design is adopted in this method is:
2. Rift Valley fever virus fluorescence quantitative PCR detection novel method as claimed in claim 1 is characterized in that this method is specially:
1) design primer probe;
2) select fluorescein according to instrument;
3) synthetic primer and probe;
4) preparation positive plasmid standard substance;
5) operation PCR;
6) data analysis;
7) extract viral RNA and carry out the system checking.
3. Rift Valley fever virus fluorescence quantitative PCR detection novel method as claimed in claim 2, it is characterized in that, described step 2) the selective fluorescent emission group of fluorescein has 6-FAM, TET, HEX, JOE, CY3, CY5, TAMRA, Texas Red in, and the fluorescent quenching group has BHQ-1, BHQ-2, Lowa BlackTMRQ, Lowa BlackTMFQ, Dabcyl.
4. Rift Valley fever virus fluorescence quantitative PCR detection novel method as claimed in claim 2, it is characterized in that, take the gene synthesis mode to make positive plasmid CBG234-3 as positive in the described step 4): to carry out gene synthetic with prolonging 10～50bp before and after the high conserved region territory bp18-bp122 that filters out in the design of primers process respectively, be cloned into then in the pMD19-T carrier, be described positive plasmid sample, numbering CBG234-3.
5. Rift Valley fever virus fluorescence quantitative PCR detection novel method as claimed in claim 2 is characterized in that, the quantitative fluorescent PCR reaction is applicable to all quantitative fluorescent PCR reaction instrument in the described step 5).
6. Rift Valley fever virus fluorescence quantitative PCR detection novel method as claimed in claim 5, it is characterized in that described quantitative real time PCR Instrument comprises SmartCyclerII, ABI real-time PCR system, BioRad PCR in real time detection system, Stratagene quantitative polumerase chain reaction instrument.
7. Rift Valley fever virus fluorescence quantitative PCR detection novel method as claimed in claim 1 is characterized in that best amplification condition is in the described step 5):
8. a Rift Valley fever virus detects the PCR system, it is characterized in that, this system comprises that primer or other can combine the primer that is equal to of also amplification with Rift Valley fever full length sequence specificity, and its similar homology can not surpass 90%, and primer sequence is as follows:
9. a Rift Valley fever virus detects the PCR system, it is characterized in that, this system comprise primer and can with Rift Valley fever full length sequence specific probe, probe sequence is as follows:
Can combine the probe that is equal to of also amplification with Rift Valley fever full length sequence specificity, the fraction of coverage of its site areas can not surpass 50%, and its homology can not surpass 70%.
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| CN102433392A (en) * | 2011-12-13 | 2012-05-02 | 中国检验检疫科学研究院 | Primers and probe for detecting fragment S of rift valley fever virus |
| CN102719557A (en) * | 2011-12-13 | 2012-10-10 | 广东出入境检验检疫局检验检疫技术中心 | Multiplex fluorescent polymerase chain reaction (PCR) kit and primers for detecting Ebola viruses, Marburg viruses, Lassa viruses and Rift Valley fever viruses |
| CN103952494A (en) * | 2014-03-06 | 2014-07-30 | 中国动物卫生与流行病学中心 | Rift valley fever virus RT-LAMP (reverse transcription-loop-mediated isothermal amplification) detection method |
| CN110295254A (en) * | 2019-07-19 | 2019-10-01 | 江苏省农业科学院 | Identify the Multiplex real-time PCR primer and probe of detection Rift Valley fever virus |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104388584A (en) * | 2014-10-30 | 2015-03-04 | 薛芳 | Rift Valley fever virus nucleic acid molecule characteristic standard sample and preparation method thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102433392A (en) * | 2011-12-13 | 2012-05-02 | 中国检验检疫科学研究院 | Primers and probe for detecting fragment S of rift valley fever virus |
| CN102719557A (en) * | 2011-12-13 | 2012-10-10 | 广东出入境检验检疫局检验检疫技术中心 | Multiplex fluorescent polymerase chain reaction (PCR) kit and primers for detecting Ebola viruses, Marburg viruses, Lassa viruses and Rift Valley fever viruses |
| CN102433392B (en) * | 2011-12-13 | 2013-05-15 | 中国检验检疫科学研究院 | Primers and probe for detecting fragment S of rift valley fever virus |
| CN102719557B (en) * | 2011-12-13 | 2014-04-02 | 广东出入境检验检疫局检验检疫技术中心 | Multiplex fluorescent polymerase chain reaction (PCR) kit and primers for detecting Ebola viruses, Marburg viruses, Lassa viruses and Rift Valley fever viruses |
| CN103952494A (en) * | 2014-03-06 | 2014-07-30 | 中国动物卫生与流行病学中心 | Rift valley fever virus RT-LAMP (reverse transcription-loop-mediated isothermal amplification) detection method |
| CN110295254A (en) * | 2019-07-19 | 2019-10-01 | 江苏省农业科学院 | Identify the Multiplex real-time PCR primer and probe of detection Rift Valley fever virus |
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