CN108018378A - A kind of Luohu virus Taq-man fluorescence probe quantitative PCRs detection kit and detection method - Google Patents
A kind of Luohu virus Taq-man fluorescence probe quantitative PCRs detection kit and detection method Download PDFInfo
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Abstract
The present invention relates to a kind of Luohu FLuorescent quantitative PCR detecting reagent box, including RT PCR reaction solutions, Taq enzyme, reverse transcriptase, Luohu FLuorescent quantitative reaction solution and the deionized water without RNase；The Luohu FLuorescent quantitative reaction solution includes two primers for detecting Luohu virus and the probe of a detection Luohu virus, and the probe of the detection Luohu virus is marked with fluorescent reporter group at its 5' end, and 3' ends are marked with fluorescent quenching group.Detection kit (1) of the present invention have height specificity, it can only amplify the nucleic acid of Luohu virus, without with other etiology nucleic acids generation cross reaction the characteristics of；(2) sensitivity of the diagnosis with height for Luohu virus；(3) testing result accuracy is high, and detection speed is fast.
Description
Technical field
The present invention relates to fishes virus vitro detection technology, more particularly to Luohu FLuorescent quantitative PCR detecting reagent box
And detection method, it belongs to viral molecular biology field.
Background technology
From 2000 so far, China's Tilapia mossambica cultivation amount ranks first in the world always, in recent years, China's Tilapia mossambica cultured output
Growing trend is still kept, about 1,780,000 t of 2015 gross annual output amount, accounts for the 30% of global total output.Tilapia mossambica industry goes out as China
Mouth guidance type industry, relies primarily on the export trade to maintain industry to operate.The country shares 133 Tilapia mossambica export processing enterprises,
About 400,000 tons of total export volume, sells to 97 countries and regions.The export volumes for starting China Tilapia mossambica in 2002 and export amount are basic
All it is in the situation of sustainable growth, Tilapia mossambica export volume in 2005 is 10.74 ten thousand tons, and 2009 are 25.89 ten thousand tons, are within 2013
40.67 ten thousand tons, all-time high is created, general export volume is 15.13 hundred million dollars.Rofe fish culture is earned foreign exchange as China's export
With the important channel of fisherman's increased income.However, in recent years the state such as Israel, Ecuador and Egypt break out in succession it is related with virus
Cultivation and wild Tilapia mossambica mortality event, the Tilapia mossambica aquaculture national to these cause heavy losses.Through research
It was found that the cause of disease for causing these dead fish events is that Luohu is viral (Tilapia lake virus, TiLV), it is a kind of Novel fish
RNA virus.Viral nucleic acid is found in the liver of sick fish, encephalopathic stove, observe at the same time from Israel and Ecuador's dead fish event
After being bred in cultivating cell, after being inoculated into healthy fish body, same symptoms can be produced to by the TiLV that sick fish tissues are separated to
Disease, prompt the cause of disease that TiLV is Israel and Ecuador's dead fish event.Israel is adjacent with Egyptian two countries, but another
Geographically being separated by very remote Ecuador of South American nations, also there occurs dead fish thing is caused by virus of the same race, also there occurs led by TiLV
The extensive dead fish event caused, illustrates that the virus causes threat to global Tilapia mossambica aquaculture.
Luohu virosis is a kind of highly pathogenic and Tilapia mossambica virosis of high mortality emerging in recent years, and cause of disease is
Luohu virus (Tilapia lake virus, TiLV), infects the viral Tilapia mossambica death rate and is up to more than 90%, TiLV and be
Just viscous sample Viraceae, minus-stranded rna virus, genome are divided into 10 segments.Untill up to now, Ecuador, Israel, brother's human relations
Than multiple countries and regions such as Asia, Egypt, Thailand report that thus disease is broken out, although China has no Luohu virosis at present
Relevant report, but the Tilapia mossambica disease broken out more, its symptom and Luohu virosis and its similar, and detected TiLV,
But the popularity of domestic Luohu virus is also totally unknown.For the detection method of TiLV, there are nido and semi-nested RT-PCR, and
In situ hybridization, these detection method operating process are complex, and time-consuming, it is necessary to specific instrument and equipment, and nido and half
The false positive rate of RT-Nested PCR is higher, and therefore, these existing detection methods are not all suitable for the efficient, fast of Luohu virus
Speed detection, it is even more impossible to carry out quantitative analysis to virus.
Real-Time Fluorescent Quantitative PCR Technique (Real-time quantitative Polymerase Chain Reaction,
Real TimePCR) it is the nucleic acid quantitation technique to grow up in qualitative PCR technical foundation.Real-Time Fluorescent Quantitative PCR Technique
Released in 1996 by Applied biosystems companies of the U.S., add fluorophor in PCR reaction systems, utilize fluorescence
Signal accumulation monitors whole PCR processes in real time, each circulation is become " visible ", finally by Ct values and standard curve to sample
The initial concentration of DNA (cDNA) in product carries out quantitative method.If detected for RNA, this is referred to as the real-time PCR of reverse transcription
I.e. (Real-time RT-PCR) is real-time PCR methodology, it refers to that the RNA to DNA or by reverse transcription (RT-PCR) passes through polymerization
The amplification process of enzyme chain reaction and real time monitoring of DNA, amplified production is just measured in the Exponential growth stage of amplification, because amplification refers to
Rise period measured value is counted with specific DNA (RNA) initial amount there are correlation, so as to fulfill quantitative detection.RealTime PCR's
Elementary object is accurate measurement and differentiates very micro specific nucleic acid, so as to be realized by monitoring CT values to original mesh
Mark the content quantitative of gene.Quantitative real-time PCR biggest advantage is to overcome terminal PCR methods into plateau or cry full
With large error quantitative after the phase, the accurate quantification of DNA/RNA is realized.The technology, which not only realizes, determines DNA/RNA templates
Amount, and with sensitivity and specificity it is high, can realize that multiple reaction, the degree of automation are high, pollution-free, real-time and accurate etc. special
Point.In addition, by adding two or more than two primers and corresponding probe in the system, can realize to a variety of cause of diseases
Detected while body.
Therefore, the technology that TiLV fluorescent quantitations quickly detect is established, can not only accomplish that carrying out discriminating to Luohu virus examines
Disconnected and quantitative analysis, and can simplify procedures, is cost-effective.Epidemiology survey, cause of disease for Luohu virosis
Monitoring and early warning all have great importance.
The content of the invention
The main object of the present invention is to provide a kind of can detect at the same time and is examined with the quantitative fluorescent PCR of quantitative analysis Luohu virus
Test agent box and detection method, the kit and detection method sensitiveness are strong, and specificity is high, can be fast and accurately to TiLV
Diagnosed, quantitative analysis and Pathogen identification.
In order to solve the above technical problems, the present invention uses following technical scheme：
A kind of Luohu FLuorescent quantitative PCR detecting reagent box, including RT-PCR reaction solutions, Taq enzyme, reverse transcriptase, sieve
Lake FLuorescent quantitative reaction solution and the deionized water without RNase；Wherein,
The Luohu FLuorescent quantitative reaction solution includes the primer and a detection Luohu of two detection Luohu virus
The probe of virus, the sequence of the primer of described two detection Luohu virus are respectively：
Sense primer TiLV-PF Seq No.1：5'GAC TGC AGC TAT GTT ATC TGG 3'；
Anti-sense primer TiLV-PR Seq No.2：5'TGG TGT AAG TGG GGT TGT T 3'；
The sequence of the probe of the detection Luohu virus is as follows：
Probe TiLV-P Seq No.3：5'AAG CAG CAG GAA TGT GCC TAT3'；
The probe of the detection Luohu virus is marked with fluorescent reporter group at its 5' end, and 3' ends are marked with fluorescent quenching
Group.
Further, the Luohu FLuorescent quantitative PCR detecting reagent box includes 12.5 μ L of RT-PCR reaction solutions,
0.5 μ L of Taq enzyme, 0.5 μ L of reverse transcriptase, 4 μ L of Luohu FLuorescent quantitative reaction solution, the 2.5 μ L of deionized water of no RNase.
Further, the Luohu FLuorescent quantitative reaction solution is by the 100uM sense primers TiLV-PF of 5 μ L, 5 μ L
100uM anti-sense primers TiLV-PR and 4 μ L 100uM probe primers TiLV-P add 169 deionized waters of the μ L without RNase
In be made.
The present invention tests the ratio of above-mentioned primer and probe, between them different ratios for
The specificity and sensitivity of testing result have significant difference, when above-mentioned primer and probe is under following proportionings, with optimal
Detection result：Detect Luohu virus primer and probe between proportioning be respectively：Seq No.1：Seq No.2：Seq
No.3=5：5：4.
Further, the fluorescent reporter group is FAM, HEX, ROX, CY3 or CY5；The fluorescent quenching group
For TAMRA, BHQ1, BHQ2 or BHQ3.
Further, the reverse transcriptase is M-MLV reverse transcriptases, and the Taq enzyme is hot start Taq polymerase.
Positive control reference in the Luohu FLuorescent quantitative PCR detecting reagent box of the present invention also containing Luohu virus
Product：The nutrient solution of TiLV-ZJ1710 plants of the Luohu virus of inactivation.
Also contain negative control reference material in the Luohu FLuorescent quantitative PCR detecting reagent box of the present invention：No RNA's goes
Ionized water.
The invention also provides a kind of Luohu virus fluorescence quantitative PCR detection method, include the following steps：
S1：Extract sample to be tested fish RNA；
S2：Preparation of reagents
Following component is put into each PCR reaction tubes：
Wherein, the Luohu FLuorescent quantitative reaction solution includes two primers for detecting Luohu virus and a detection
The probe of Luohu virus, and Luohu FLuorescent quantitative reaction solution is configured according to following table：
| Title | Addition (μ L) |
| 100uM TiLV-PF | 5 |
| 100uM TiLV-PR | 5 |
| 100uM TiLV-P | 4 |
| Without RNase deionized water | 169 |
The sequence of the primer of described two detections Luohu virus is respectively：
TiLV-PF Seq No.1：5'GAC TGC AGC TAT GTT ATC TGG 3'；
TiLV-PR Seq No.2：5'TGG TGT AAG TGG GGT TGT T 3'；
The sequence of the probe of the detection Luohu virus is as follows：
TiLV-P Seq No.3：5'AAG CAG CAG GAA TGT GCC TAT3'；
S3：Fluorescence quantitative RT-RCR expands；
S4：Interpretation of result
Fluorescence curve is in " S " type curve and CT≤34.0 in FAM passages, is judged as Luohu virus-positive；Without typical " S "
Type expands or CT>34.0, it is judged as that Luohu virus is negative.
Preferably, the condition of fluorescence quantitative RT-RCR is in the S3：First 42 DEG C of 30min, 95 DEG C of 5min are carried out inverse
Transcription, then 95 DEG C of 8s, 58 DEG C of 35s carry out 40 circulations.
The present invention have the advantage that compared with prior art for：
(1) detection kit of the present invention have height specificity, it can only amplify the nucleic acid of Luohu virus, without with
Cross reaction occurs for other etiology nucleic acids；
(2) diagnosis sensitivity with height of the kit of the present invention for Luohu virus；
(3) kit testing result accuracy of the invention is high, and detection speed is fast.
Brief description of the drawings
Fig. 1 is Luohu virus real-time fluorescence quantitative PCR canonical plotting.
Fig. 2 is the specific test result of kit detection Luohu virus of the present invention；Wherein mark 1 for positive control,
Be the amplification curve using TiLV nucleic acid as template labeled as 2, mark 3-9 be with Streptococcusagalactiae, Aeromonas hydrophila,
KHV, IHNV, LMBV, ISKNV and GCRV nucleic acid are the amplification curve of template, are negative control labeled as 10.
Fig. 3 is the sensitivity test result of kit detection Luohu virus of the present invention；It is positive right wherein labeled as 1
According to comparing and dilute 10 respectively for 2-9 for the RNA of TiLV1To 108It is negative right labeled as 10 for the amplification curve of template
According to.
Fig. 4 is the result of the test of kit detection detection Luohu virosis clinical sample of the present invention；Wherein mark 1 for sun
Property control, labeled as 2 and 3 be TiLV detect positive, mark 4-9 for TiLV detect negative sample, labeled as 10
For negative control.
Embodiment
The present invention is further described with reference to specific implementation case, the advantages and features of the present invention will be with describing
It is and apparent.But these case study on implementation be full of it is exemplary, not to the scope of the present invention form any restrictions.This area skill
Art personnel should be understood that without departing from the spirit and scope of the invention can be to the details of any technical solution of the present invention
Modify or replace with form, but these modifications and replacement are each fallen within protection scope of the present invention.
Embodiment 1：The design and synthesis of specific primer
TiLV-AD-2016, TiLV-4-2011, TiLV-TV1, TiLV-TV2, TiLV-TV3, TiLV- are obtained from NCBI
TV4, TiLV-TV5, TiLV-TV6, TiLV-TV7 and this laboratory be isolated from the S1 gene orders of two plants of TiLV in Guangdong into
Row compares, analysis, using 6.0 softwares of Oligo, according to these Luohu virus isolated strain S1 gene 5' ends and 3' ends conservative region
Design primer and probe.Sense primer TiLV-PF sequences are：5'GAC TGC AGC TAT GTT ATC TGG 3', downstream is drawn
Thing TiLV-PR sequences are：5'TGG TGT AAG TGG GGT TGT T 3', the report of probe TiLV-P sequences and both ends mark
Group and quenching group are：FAM-AAG CAG CAG GAA TGT GCC TAT-TAMRA.
Embodiment 2：The foundation of Luohu virus fluorescence quantitative PCR detection method standard curve
The extraction of S1, viral RNA
(Invitrogen companies, article No. are purchased from by Trizol kits：Specification 15596-026) or according to Qiagen
RNA extracts kits (are purchased from Qiagen companies, article No.：74104) specification step, from the fish body internal organ of the corresponding virus of infection
RNA is extracted in tissue, the template as RT-PCR reactions.The key step of Trizol methods extraction total serum IgE is as follows：Take tissue sample
500 μ L Trizol reagents are added, 5 minutes are stood after mixing, add 400 μ L chloroforms, vibration mixes 1min, and 12000rpm is in 4 DEG C
15min is centrifuged, takes supernatant to add isometric isopropanol, overturns and mixes, 4 DEG C of placements 15min, 12000rpm are in 4 DEG C of centrifugations
15min, abandons supernatant, washes precipitation with 70% ethanol, 12000rpm centrifuges 5min in 4 DEG C, is resuspended and precipitated without RNAse water with 30 μ L.
S2, preparation of reagents
Component detection reagent as shown in table 1 and RNA samples are put into each PCR reaction tubes：
Table 1
Wherein, the Luohu FLuorescent quantitative reaction solution includes two primers for detecting Luohu virus and a detection
The probe of Luohu virus, and Luohu FLuorescent quantitative reaction solution is configured according to table 2 below：
Table 2
| Title | Addition (μ L) |
| 100uM TiLV-PF | 5 |
| 100uM TiLV-PR | 5 |
| 100uM TiLV-P | 4 |
| Without RNase deionized water | 169 |
S3, fluorescence quantitative RT-RCR amplification
Fluorescence curve is in " S " type curve and CT≤34.0 in FAM passages, is judged as Luohu virus-positive；Without typical " S "
Type expands or CT>34.0, it is judged as that Luohu virus is negative.
The Luohu FLuorescent quantitative standard curve the result is shown in Figure 1 of foundation.
Embodiment 3：Luohu virus fluorescent quantitative RT-PCR kit specific test
In order to detect the specificity of kit of the present invention, with the present invention Luohu virus fluorescent quantitative RT-PCR kit come
Detect Streptococcusagalactiae, Aeromonas hydrophila, that TiLV, KHV, IHNV, LMBV, ISKNV, GCRV analyze this method is other to fish
Encountered pathogenic and Luohu viral diagnosis situation, while with the nutrient solution of TiLV-ZJ1710 plants of the Luohu virus of inactivation and without RNA's
Deionized water sets up positive control and negative control.
Testing result shows：Positive control has amplified signal, and negative control does not have amplified signal, and FAM passages are only to sieve
Lake virus is expanded, and sees Fig. 2, and does not have amplified signal by the use of the nucleic acid of other cause of diseases as passage during template.Show this hair
Bright detection kit can only specific amplification go out the nucleic acid of Luohu virus, without cross reaction occurs with other etiology nucleic acids.
Embodiment 4：Luohu virus fluorescent quantitative RT-PCR kit sensitivity tests
This experiment carries out 10 times of doubling dilutions to Luohu viral RNA, and testing result shows, kit has well sensitive
Property, and CT values are reduced with concentration and changed in gradient, see Fig. 3.Result of the test shows that diagnosis of the kit for Luohu virus has
There is the sensitivity of height, while set up with the nutrient solution of TiLV-ZJ1710 plants of Luohu virus of inactivation and the deionized water without RNA
Positive control and negative control.
Embodiment 5：Luohu virus fluorescent quantitative RT-PCR kit detects clinical sample
The clinical 8 parts of Tilapia mossambica samples obtained are chosen using PCR method and cell separation to be detected, while to go out
The nutrient solution of TiLV-ZJ1710 plants of Luohu virus and the deionized water without RNA living sets up positive control and negative control.Such as figure
Shown in 4, positive control has amplified signal, and negative control does not have amplified signal, in the RNA of this 8 parts of sample extractions, PCR amplification
Separated with cell and verify that wherein identical 2 parts of samples are the positive, prompt infection of this 2 parts of samples there are TiLV, and other 6 parts
Sample is then feminine gender.Using the Luohu virus fluorescent quantitative RT-PCR kit of foundation, 8 parts of Tilapia mossambica samples are examined
Survey, the results show that PCR method and cell separation are accredited as 2 parts of samples of the positive, fluorogenic quantitative detection is also the positive, such as Fig. 4 institutes
Show, prompt the Luohu virus fluorescent quantitative RT-PCR kit that the present invention develops to clinical sample detection and virus purification and PCR
Detection method has good uniformity.
Embodiment described above only expresses some embodiments of the present invention, its description is more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect scope.
Claims (9)
1. a kind of Luohu FLuorescent quantitative PCR detecting reagent box, it is characterised in that including RT-PCR reaction solutions, Taq enzyme, inverse
Transcriptase, Luohu FLuorescent quantitative reaction solution and the deionized water without RNase；Wherein,
The Luohu FLuorescent quantitative reaction solution includes the primer and a detection Luohu virus of two detection Luohu virus
Probe, the sequence of primers of described two detection Luohu viruses is respectively：
Sense primer TiLV-PF Seq No.1：5'GAC TGC AGC TAT GTT ATC TGG 3'；
Anti-sense primer TiLV-PR Seq No.2：5'TGG TGT AAG TGG GGT TGT T 3'；
The sequence of the probe of the detection Luohu virus is as follows：
Probe TiLV-P Seq No.3：5'AAG CAG CAG GAA TGT GCC TAT3'；
The probe of the detection Luohu virus is marked with fluorescent reporter group at its 5' end, and 3' ends are marked with fluorescent quenching base
Group.
2. kit according to claim 1, it is characterised in that the Luohu FLuorescent quantitative PCR detecting reagent box
Include 12.5 μ L of RT-PCR reaction solutions, 0.5 μ L of Taq enzyme, 0.5 μ L of reverse transcriptase, 4 μ L of Luohu FLuorescent quantitative reaction solution,
2.5 μ L of deionized water without RNase.
3. kit according to claim 1 or 2, it is characterised in that the Luohu FLuorescent quantitative reaction solution is by 5
The 100uM probe primers TiLV- of the 100uM sense primers TiLV-PF of μ L, the 100uM anti-sense primers TiLV-PR of 5 μ L and 4 μ L
P is added in 169 deionized waters of the μ L without RNase and is made.
4. kit according to claim 1, it is characterised in that the fluorescent reporter group for FAM, HEX, ROX,
CY3 or CY5；The fluorescent quenching group is TAMRA, BHQ1, BHQ2 or BHQ3.
5. kit according to claim 1, it is characterised in that the reverse transcriptase is M-MLV reverse transcriptases, described
Taq enzyme be hot start Taq polymerase.
6. kit according to claim 1, it is characterised in that the positive control ginseng in kit also containing Luohu virus
Examine product：The nutrient solution of TiLV-ZJ1710 plants of the Luohu virus of inactivation.
7. kit according to claim 1, it is characterised in that also contain negative control reference material in kit：Without RNA
Deionized water.
8. a kind of Luohu virus fluorescence quantitative PCR detection method, it is characterised in that include the following steps：
S1：Extract sample to be tested fish RNA；
S2：Preparation of reagents
Following component is put into each PCR reaction tubes：
Wherein, the Luohu FLuorescent quantitative reaction solution includes the primer and a detection Luohu of two detection Luohu virus
The probe of virus, and Luohu FLuorescent quantitative reaction solution is configured according to following table：
The sequence of the primer of described two detections Luohu virus is respectively：
TiLV-PF Seq No.1：5'GAC TGC AGC TAT GTT ATC TGG 3'；
TiLV-PR Seq No.2：5'TGG TGT AAG TGG GGT TGT T 3'；
The sequence of the probe of the detection Luohu virus is as follows：
TiLV-P Seq No.3：5'AAG CAG CAG GAA TGT GCC TAT3'；
S3：Fluorescence quantitative RT-RCR expands；
S4：Interpretation of result
Fluorescence curve is in " S " type curve and CT≤34.0 in FAM passages, is judged as Luohu virus-positive；Expand without typical " S " type
Increasing or CT>34.0, it is judged as that Luohu virus is negative.
9. according to the method described in claim 8, the condition of fluorescence quantitative RT-RCR is in the S3：First 42 DEG C of 30min, 95 DEG C
5min carries out reverse transcription, and then 95 DEG C of 8s, 58 DEG C of 35s carry out 40 circulations.
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| CN111004865A (en) * | 2018-10-05 | 2020-04-14 | 福又达生物科技股份有限公司 | Method for detecting fish pathogens |
| TWI730432B (en) * | 2018-10-05 | 2021-06-11 | 福又達生物科技股份有限公司 | Methods for detecting pathogens in fish |
| CN110616281A (en) * | 2019-10-29 | 2019-12-27 | 周伟光 | Method for quantitatively detecting Luo lake virus by using real-time fluorescent RT-PCR |
| CN112458203A (en) * | 2020-10-27 | 2021-03-09 | 深圳市莱孚生物科技有限公司 | Primer, probe and detection method for identifying Luo lake virus |
| CN113755645A (en) * | 2021-09-30 | 2021-12-07 | 厦门海关技术中心 | Fluorescent quantitative RT-PCR primer pair and probe for detecting Luo lake virus, kit and detection method |
| CN113755645B (en) * | 2021-09-30 | 2023-06-09 | 厦门海关技术中心 | Fluorescent quantitative RT-PCR primer pair and probe for detecting rochu virus, kit and detection method |
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Application publication date: 20180511 |