CN102268487B - Dual fluorescence PCR detection kit for dengue virus III/IV - Google Patents
Dual fluorescence PCR detection kit for dengue virus III/IV Download PDFInfo
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Abstract
The invention discloses a dual-fluorescence PCR detection kit for dengue virus III/IV, belonging to the field of detection of dengue virus. The kit comprises the following components: RT-PCR reaction liquid, enzyme mixed liquid, dengue virus type III/type IV double reaction liquid and RNase removing water. The dual-fluorescence PCR detection kit for dengue virus III/IV solves the problem that the existing in-vitro detection kit cannot simultaneously detect different subtypes of dengue virus in the same reaction tube, can simultaneously detect dengue virus III and IV in the same reaction tube, and has the advantages of strong specificity, high sensitivity, simple and convenient operation, strong repeatability, rapid and objective detection result and the like.
Description
Technical field
The present invention relates to a kind of dengue fever virus vitro detection test kit, relate in particular to dengue fever virus III type/IV type double fluorescent PCR detection kit, the invention belongs to the vitro detection field of dengue virus serotype type.
Background technology
Singapore hemorrhagic fever (Classical dengue fever, DF) be a kind of acute infectious disease that 4 serotypes by dengue fever virus (dengue virus) cause, mainly propagate by Aedes aegypti and Aedes albopictus, clinically take high hot, hemorrhage, whole-body muscle and arthrodynia etc. as cardinal symptom, can be with fash, lymphadenopathy and oligoleukocythemia.Dengue virus infection is except causing singapore hemorrhagic fever, and serious also can cause dengue hemorrhagic fever (denguehemorrhagic fever, DHF) or step on symptoms such as removing from office shock syndromes (dengue shocksyndrome, DSS).
DHF is comparatively serious a kind of Clinical types take high hot, hemorrhage, shock and high case fatality rate as feature.Because global warming, international tourism frequently and to mosquito lack effectively control, singapore hemorrhagic fever worldwide occured repeatedly to be very popular, the whole world has more than 100 country to occur popular, add up according to WHO, annual dengue infection number has 5,000 ten thousand, the whole world has 2,500,000,000 people just being infected the threat of dengue virus, and every average annual millions of cases that occur are comparatively serious public health problems.
Dengue virus is the important member that flaviviridae (flaviviridae) Flavivirus (flavivirus) is stepped on the leather subgroup, and the dengue virus that occurring in nature exists can be divided into 4 serotypes.The contagium of singapore hemorrhagic fever mainly is the dominant and recessive the infected who is in the viremia phase, is main source of infection urban type plague area district patient and inapparent infection person.At the general susceptible in new epidemic-stricken area.But the highest with between twenty and fifty sickness rate.At the region Endemic Area, the resident below 20 years old, 100% can detect the neutralizing antibody of anti-dengue virus in serum, thereby a patient mostly is children.
The history in existing more than 200 year of the discovery of singapore hemorrhagic fever, the report of early stage record is Cairo, EGY, Indonesia Jakarta and U.S. Philadelphia in 1779, and according to symptom called after joint heat and fracture fever.1869 by London Society of Internal Medicine of imperial family called after singapore hemorrhagic fever.China is popular in Guangdong in 1978, and isolates IV type dengue fever virus.After this, 1979,1980,1985 little I type, II, the III C-type virus Cs isolated in the groove.At present each department are mainly broken out or are dissipated as the master to be dispersed in, and with imported case cause popular in the majority.
Real-Time Fluorescent Quantitative PCR Technique (Real-time quantitative Polymerase Chain Reaction is called for short Real Time PCR) is the nucleic acid quantification technology that grows up in the qualitative PCR technical foundation.Real-Time Fluorescent Quantitative PCR Technique was released by U.S. Applied biosystems company in 1996, in the PCR reaction system, add fluorophor, utilize the whole PCR process of fluorescent signal accumulation Real-Time Monitoring, each circulation is become " as seen ", by Ct value and typical curve the initial concentration of the DNA in the sample (cDNA) is carried out quantitative method at last.If being used for RNA detects, this is called as the reverse transcription PCR in real time is that (Real-time RT-PCR) is the PCR in real time method, it refers to that the RNA to DNA or process reverse transcription (RT-PCR) passes through the amplification process of polymerase chain reaction and real time monitoring of DNA, Exponential growth stage in amplification is just measured amplified production, because there are dependency in augmentation index rise period observed value and specific DNA (RNA) initial amount, thereby realize detection by quantitative.The elementary object of RealTime PCR is accurately to measure and differentiate very micro-specific nucleic acid, thereby can realize content quantitative to the original object gene by monitoring CT value.The advantage of real-time fluorescence quantitative PCR method maximum is quantitative larger error after having overcome terminal point PCR method and entering plateau or the period of saturation of crying, and realizes the accurate quantification of DNA/RNA.This technology not only realized to the DNA/RNA template quantitatively, and have susceptibility and specificity is high, can realize multiple reaction, level of automation is high, pollution-free, real-time and the characteristics such as accurate.In addition, by in this system, adding two or two above primers and corresponding probe, detect when just can realize multiple pathogens.
Existing dengue fever virus external diagnosis reagent case scarcely can simultaneously detect respectively dengue fever virus III type and IV type in a reaction tubes, and has specificity and the defective such as susceptibility is lower, detecting step is loaded down with trivial details, haves much room for improvement.
Summary of the invention
It is a kind of double fluorescent PCR detection kit that can detect respectively dengue fever virus III type, IV type simultaneously in a reaction tubes that main purpose of the present invention provides, and has high specificity, susceptibility advantages of higher.
In order to achieve the above object, the present invention has adopted such technical scheme:
A kind of dengue fever virus III type/IV type double fluorescent PCR detection kit comprises following each component: RT-PCR reaction solution, enzyme mixation, dengue fever virus III type/IV double reaction liquid and remove RNA enzyme water.
Wherein, described dengue fever virus III type/IV type double reaction liquid is comprised of following 2 groups of components:
Component (1): primer and a probe that detects dengue fever virus III type by a pair of detection dengue fever virus III type form; Wherein, the base sequence of two primers is respectively shown in SEQ ID No.1 and the SEQ ID No.2; The base sequence of probe is shown in the SEQ ID No.3, and 5 of this probe ' end is marked with the fluorescence report group, and 3 ' end is marked with the fluorescent quenching group.
Component (2): primer and a probe that detects dengue fever virus IV type by a pair of detection dengue fever virus IV type form; Wherein, the base sequence of two primers is respectively shown in SEQ ID No.4 and the SEQ IDNo.5; The base sequence of probe is shown in the SEQ ID No.6, and 5 of this probe ' end is marked with the fluorescence report group, and 3 ' end is marked with the fluorescent quenching group.
Wherein, described fluorescence report group comprises the fluorescence report groups such as FAM, HEX, ROX, CY3 or CY5; Described fluorescent quenching group comprises the fluorescent quenching groups such as BHQ1, BHQ2 or BHQ3;
The present invention gropes the proportioning ratio of above-mentioned primer and probe and optimizes, test-results is found, different proportioning ratio has significant difference for specificity and the susceptibility of detected result between them, the present invention finds by high-throughout shaker test, above-mentioned primer and probe have optimum detection effect under following proportioning:
Detecting the primer of dengue fever virus III type and the proportioning of probe is respectively: SEQ ID No.1: SEQ ID No.2: SEQ ID No.3=500nM: 500nM: 400nM;
Detecting the primer of dengue fever virus IV type and the proportioning of probe is respectively: SEQ ID No.4: SEQ ID No.5: SEQ ID No.6=600nM: 600nM: 300nM.
Table 1 dengue fever virus III type/IV type primer probe
Table 2 dengue fever virus III type/IV type double reaction fluid component
| Reagent name | Add volume (μ l)/50 reaction |
| Dengue fever virus III type FP (SEQ ID No.1) | 6.25 |
| Dengue fever virus III type RP (SEQ ID No.2) | 6.25 |
| Dengue fever virus III type P (SEQ ID No.3) | 5 |
| Dengue fever virus IV type FP (SEQ ID No.4) | 7.5 |
| Dengue fever virus IV type RP (SEQ ID No.5) | 7.5 |
| Dengue fever virus IV type P (SEQ ID No.6) | 3.75 |
In order to reach better detection effect, also can contain the positive reference material (available from Beijing three rich polygala root bio tech ltd or Shanghai Jierui Biology Engineering Co., Ltd) of dengue fever virus III type/IV type in the test kit of the present invention.
Described enzyme mixation comprises RNA enzyme inhibitors, Taq enzyme, reversed transcriptive enzyme, 10 * damping fluid, removes RNA enzyme water.Reversed transcriptive enzyme and archaeal dna polymerase play very important effect in pcr amplification, comprise efficient and the specificity of reverse transcription, amplification, and in order to reach better effect, the present invention preferably adopts M-MLV reversed transcriptive enzyme and warm start enzyme.
Another object of the present invention provides the using method that test kit of the present invention detects dengue fever virus III type/IV type, comprising:
(1) RNA of extraction sample;
(2) prepare following component: RT-PCR reaction solution 12.5ul, enzyme mixation 1ul, dengue fever virus III type/IV type reaction solution 4ul removes RNA enzyme water 2.5ul, RNA sample 5ul.
(3) RT-PCR amplification: carry out the RT-PCR amplification according to following program
(4) result judges: fluorescence curve is " S " type curve and CT≤35.0 in the FAM passage, and it is positive to be judged as dengue fever virus III type; Without the amplification of typical case's " S " type or CT＞35.0, it is negative to be judged as dengue fever virus III type.Fluorescence curve is " S " type curve and CT≤35.0 in the VIC passage, and it is positive to be judged as dengue fever virus IV type; Without the amplification of typical case's " S " type or CT＞35.0, it is negative to be judged as dengue fever virus IV type.
Before the detection, first according to the RNA extracting method of routine to sample extraction RNA, sample to be measured is added in the PCR reaction tubes of ready dengue fever virus III type/IV type reagent augmentation detection in the fluorescent quantitative PCR instrument.After amplification finishes, judge whether to infect leather fever virus III type or IV type according to fluorescence curve.
Dengue fever virus III type of the present invention/IV type double fluorescent PCR detection kit can detect dengue fever virus III type and IV type simultaneously, solved currently available products can only detect a kind of hypotype dengue fever virus in a tube reaction problem.That the present invention also has is easy and simple to handle, repeatable strong, the advantage such as detected result is quick and objective, has extremely wide application prospect in singapore hemorrhagic fever in-vitro diagnosis field.
Description of drawings
Fig. 1 test kit of the present invention detects the graphic representation of five groups of samples.
Fig. 2 test kit of the present invention detects the susceptibility test-results of dengue fever virus III type.
Fig. 3 test kit of the present invention detects the susceptibility test-results of dengue fever virus IV type.
Fig. 4 test kit of the present invention detects the specific test result of dengue fever virus III type.
Fig. 5 test kit of the present invention detects the specific test result of dengue fever virus IV type.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not consisted of any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can make amendment or replace the details of technical solution of the present invention and form, but these modifications and replacing all fall within the scope of protection of the present invention.
The detection test of test example 1 dengue fever virus III type of the present invention/IV type double fluorescent PCR test kit clinical sample
1 sample process (RNA extraction)
1.1 get 200ul singapore hemorrhagic fever clinical sample, add 400ul Binding Buffer supplemented with Poly (A), fully change the High Purity strainer tube over to behind the mixing, the centrifugal 15s of 8000rmp discards the waste liquid in the collection tube.
1.2 add 500ul Inhibitor Removal Buffer to strainer tube, the centrifugal 1min of 8000rmp discards the waste liquid in the collection tube.
1.3 add 450ul Washing Buffer to strainer tube, the centrifugal 1min of 8000rmp discards the waste liquid in the collection tube.
1.4 repeating step 3, high speed centrifugation 10s then, this step must be removed waste liquid clean.
1.5 add 50ul Elution Buffer to strainer tube, room temperature leaves standstill 2min, the centrifugal 1min of 8000rmp, and centrifugal gained solution is the RNA of purifying.
2 reagent are prepared
Table 3
The 3PCR amplification program
4 interpretations of result: 5 clinical sample concrete outcomes see Table 4 and Fig. 1.
Table 4
Test example 2 dengue fever virus III types of the present invention/IV type double fluorescent PCR test kit susceptibility test
This test is carried out 10 times of doubling dilutions to dengue fever virus III type plasmid (available from Beijing three rich polygala root bio tech ltds) and dengue fever virus IV type plasmid (available from Shanghai Jierui Biology Engineering Co., Ltd), detected result shows, test kit of the present invention has good susceptibility, and the CT value reduces in gradient change (Fig. 2, Fig. 3) with concentration.Test-results shows that test kit of the present invention has the susceptibility of height for the diagnosis of dengue fever virus III type/IV type.
Test example 3 dengue fever virus III types of the present invention/IV type double fluorescent PCR test kit specific test
In order to detect the specificity of test kit of the present invention, detect dengue fever virus strain, west nile virus strain, encephalitis b virus strain, chikungunya disease poison strain, yellow fever virus strain, Syndebis virus strain etc. with dengue fever virus III type of the present invention/IV type detection kit.
Test-results shows that the FAM passage only increases (Fig. 4) to dengue fever virus III type, and the VIC passage is only to dengue fever virus IV type increase (Fig. 5).Show detection kit of the present invention can the specific amplification dengue fever virus and not with other viral nucleic acid generation cross reaction.
KLPI110307
＜110〉the large generation bio tech ltd in Jiangsu
＜120〉dengue fever virus III type/IV type double fluorescent PCR detection kit
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Claims (5)
1. dengue fever virus III type/IV type double fluorescent PCR detection kit is characterized in that, comprises following each component: RT-PCR reaction solution, enzyme mixation, dengue fever virus III type/IV type double reaction liquid and remove RNA enzyme water;
Described dengue fever virus III type/IV type double reaction liquid is comprised of following component (1) and component (2):
Component (1): primer and a probe that detects dengue fever virus III type by a pair of detection dengue fever virus III type form; Wherein, the right base sequence of described primer is respectively shown in SEQ ID No.1 and the SEQ ID No.2; The base sequence of described probe is shown in the SEQ ID No.3, and 5 of this probe ' end is marked with the fluorescence report group, and 3 ' end is marked with the fluorescent quenching group;
Component (2): primer and a probe that detects dengue fever virus IV type by a pair of detection dengue fever virus IV type form; Wherein, the right base sequence of described primer is respectively shown in SEQ ID No.4 and the SEQ ID No.5; The base sequence of described probe is shown in the SEQ ID No.6, and 5 of this probe ' end is marked with the fluorescence report group, and 3 ' end is marked with the fluorescent quenching group.
2. according to double fluorescent PCR detection kit claimed in claim 1, it is characterized in that: described fluorescence report group comprises FAM, HEX, ROX, CY3 or CY5 fluorescence report group; Described fluorescent quenching group comprises BHQ1, BHQ2 or BHQ 3 fluorescent quenching groups.
3. according to double fluorescent PCR detection kit claimed in claim 1, it is characterized in that the proportioning of each primer and probe is as follows in the component (1): SEQ ID No.1:SEQ ID No.2:SEQ ID No.3=500nM:500nM:400nM.
4. according to double fluorescent PCR detection kit claimed in claim 1, it is characterized in that the proportioning of each primer and probe is as follows in the component (2): SEQ ID No.4:SEQ ID No.5:SEQ ID No.6=600nM:600nM:300nM.
5. according to any described double fluorescent PCR detection kit of claim 1-4, it is characterized in that: the positive reference material that also contains dengue fever virus III type/IV type.
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| CN104328222B (en) * | 2014-11-17 | 2015-11-25 | 扬州大学 | The test kit of reverse transcription PCR detection and somatotype dengue virus and detection method thereof |
| CN104962661A (en) * | 2015-05-28 | 2015-10-07 | 中山大学 | RT-LAMP kit for rapidly identifying 3-type dengue viruses |
| CN108588282A (en) * | 2018-05-02 | 2018-09-28 | 北京泱深生物信息技术有限公司 | The gene diagnosis marker of dengue fever and dengue hemorrhagic fever |
| CN108531655A (en) * | 2018-05-02 | 2018-09-14 | 北京泱深生物信息技术有限公司 | Biomarker of the CIITA genes as diagnosis dengue fever and dengue hemorrhagic fever |
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| CN101139638A (en) * | 2007-05-08 | 2008-03-12 | 深圳太太基因工程有限公司 | Primer and probe sequence for detecting dengue virus 3 nucleotides fragment |
| CN102140529A (en) * | 2010-12-24 | 2011-08-03 | 中国检验检疫科学研究院 | New dengue virus fluorescence quantitative polymerase chain reaction detection method and dengue virus polymerase chain reaction detection system |
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| CN101139638A (en) * | 2007-05-08 | 2008-03-12 | 深圳太太基因工程有限公司 | Primer and probe sequence for detecting dengue virus 3 nucleotides fragment |
| CN102140529A (en) * | 2010-12-24 | 2011-08-03 | 中国检验检疫科学研究院 | New dengue virus fluorescence quantitative polymerase chain reaction detection method and dengue virus polymerase chain reaction detection system |
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