CN1786190A - Method of detecting cerebritis B virus and kit - Google Patents
Method of detecting cerebritis B virus and kit Download PDFInfo
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- CN1786190A CN1786190A CN 200410077214 CN200410077214A CN1786190A CN 1786190 A CN1786190 A CN 1786190A CN 200410077214 CN200410077214 CN 200410077214 CN 200410077214 A CN200410077214 A CN 200410077214A CN 1786190 A CN1786190 A CN 1786190A
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Abstract
The invention relates to examination clinic sample encephalitis b janpanese encephalitis JEV existing method and its kit, especially the method of using real time fixed quantity fluorescence polymerase chain reaction technique to diagnose JEV infection and its using kit.
Description
Affiliated field
The present invention relates to detect the pathogenic agent of encephalitis B in the clinical sample---method and test kit that encephalitis b virus (JEV) exists particularly relate to the method and the employed test kit that infect with real-time quantitative fluorescence polymerase chain reaction technique early diagnosis JEV.
Background of invention
Encephalitis B is a class central nervous system infection disease of propagating through mosquito bite, and its pathogenic agent is an encephalitis b virus.Encephalitis B is a kind of zoonosis, main infected pigs, horse, birds and people.Encephalitis B concentrates on the Asia in the main distribution in the whole world, some countries and regions (BurkeD.S.Monath of the torrid zone, South East Asia and subtropical zone, T.P.2001.Flaviviruses, In B.N.Fields (ed.), Fields virology, vol.1,4thed.Lippincott-Raven Publishers, Philadelphia.2.Tiroumourougane S V, Raghava P, Srinivasan S.Japanese viral encephalitis Postgrad Med J 2002; 78:205-215).Since the nineties in 20th century, there is the trend of expansion the encephalitis B plague area, certain non-epidemic regions begins to find encephalitis B popular (Zhan Ying and cleaning politics, economics, organization, and ideology, the popular overview of encephalitis and prevention and control measure, the Guangdong Prov. Disease Prevention-control Center, 03.06http: //www.cdcp.org.cn/newgc/yinao3.htm), number of the infected accounts for global more than 1/2.It is popular that 3 encephalitis B took place once in China to the seventies fifties for eighties of last century, and wherein twice case load in back is up to 150,000 and 170,000, and sickness rate is up to 20,/10 ten thousand, and case fatality rate is up to 25%.From the later stage seventies 20th century a large amount of use Vaccinum Encephalitis B since, case reduces year by year, nationwide be very popular controlled.Yet, partial area still between or encephalitis outburst or popular appears, annual case still has about 10,000～20,000.
The diagnosis of encephalitis B case mainly relies on epidemiology, Clinical symptoms and laboratory examination three aspect evidences.Laboratory examination comprises methods such as reverse passive hemagglutination suppresses that experiment, indirect immunofluorescence, enzyme-linked immunosorbent assay and virus are separated.Yet these are primarily aimed at the detection of antibodies method all can not provide strong diagnostic evidence in early days in disease.Particularly, aforesaid method all need be measured and compare acute phase and convalescent serum antibody titer, and time span is long, is unfavorable for patient's early diagnosis and therapy.Though the RT-PCR method is sensitive and can detect virus in early days, because of its complex steps and pollute easily, thereby use is restricted.
The real-time fluorescence quantitative PCR technology is to grow up based on traditional round pcr the mid-90 in last century.Compare with traditional (end point determination) round pcr, the near real-time quantitative monitoring method has not only realized the quantitative analysis of low copy number target polynucleotide, but also has specificity and advantage such as tolerance range is stronger, level of automation is higher and contamination of heavy is littler.
The real-time fluorescence PCR technology is to add fluorescence labeling probe in polymerase chain reaction (PCR) system, use a kind of pcr amplification instrument that has nuclear power coupling devices (CCD), reflect each round-robin level of amplification of PCR by the dynamic change that detects fluorescent signal in real time.CCD can periodically send the exciting light of specific wavelength according to certain procedure, collect to detect fluorescent signal, and is aggregated into workstation by software analysis and obtains amplification curve.By analysis, can carry out qualitative and quantitative analysis to test sample to amplification curve and cycle threshold (Ct).Real-time fluorescence PCR has saved the combine together whole process of detection of dynamic DNA cloning of DNA cloning and testing process the PCR last handling process and has shortened analysis time as a result greatly, makes that this method is more quick and easy.Because real-time fluorescence PCR is taked a kind of detecting pattern of sealing, aerosol pollutes and the false positive that causes thus thereby reduced.Therefore, this technology replaces traditional PCR method gradually in the detection and quantitative analysis of target polynucleotide sample, obtains very using widely.
The TaqMan round pcr is a kind of (Mackay IM et al.Real-time PCR invirology..Nucleic Acids Res.2002 5 of real-time fluorescence PCR; 30 (6): 1292-1305; Lie, Y.S., Petropoulos, C.J., Advances in quantitative PCR technology:5 ' nuclease assays.Current Opinion inBiotechnology 1998.9,43-48.).Compare with traditional PCR, it has increased the two ends probe of mark fluorescent reporter group and quenching group respectively in reaction system.When probe structure was complete, the energy that the fluorescence report group sends fluorescence was transferred to quenching group, presents quenching effect.If the existence of target sequence is arranged in the amplification procedure, the process middle probe molecule of amplification is hydrolyzed cut-out gradually, and fluorescence report group and quenching group dissociate mutually, have blocked the two FRET (fluorescence resonance energy transfer) effect, and the fluorescence report group sends fluorescent signal.Along with the carrying out of amplification, fluorescent signal presents linear the enhancing along with the segmental amplification of purpose.
FDA Food and Drug Administration (FDA) has ratified the PCR diagnostic kit of some detection by quantitative pathogenic agent, as is used for the test kit of the real-time fluorescence PCR detection of HIV, mycobacterium tuberculosis, chlamydia trachomatis etc.Equally, China at present also approved the production and the clinical application of real-time fluorescence PCR assay kit of hepatitis B, hepatitis C, HIV and SARS etc.Therefore, need a kind of real time fluorescent PCR method that can detect JEV of development now, satisfy the needs of the detection and the monitoring of encephalitis B.
As everyone knows, in using known real-time fluorescence quantitative PCR technology for detection and quantitative analysis clinical sample in the practice of certain particular target nucleic acid, in order to reduce and avoid the false negative or the false positive of detected result, improve the accuracy of quantitative analysis, critical basic fundamental link is how based on known target polynucleotide sequences Design and prepare suitable primer and oligonucleotide probe.The inventor is being engaged in round pcr particularly on the basis of real-time fluorescence quantitative PCR technical study in the past for many years, this technology is applied to the detection and the quantitative analysis of genome polynucleotide of all known varients of encephalitis b virus, has successfully finished the present invention.
The purpose of invention
An object of the present invention is to provide a kind of method of using JEV virus in the real-time fluorescence quantitative PCR technology detection by quantitative sample, this method comprises: (1) provides and comprises sample, the nucleic acid amplification system, with the fluorescence monitoring system interior reaction with detect mixture, (2) by the said target polynucleotide of amplified reaction cyclic amplification, (3) said fluorescence generation group is combined with the target polynucleotide that is amplified is indirect, (4) determine the fluorescence volume that fluorescence generation group is produced, (5) after the analysing amplified circulation, determine the existence and the relative quantity (being the copy number of target nucleic acid sequence) thereof of target polynucleotide in conjunction with the quantitative criterion curve according to the fluorescence volume that produces in the circulating reaction; Wherein the nucleic acid amplification system comprise can with target polynucleotide bonded Oligonucleolide primers, and the fluorescence real-time monitoring system comprise one can with target polynucleotide specificity bonded oligonucleotide probe; Be characterised in that employed forward and reverse oligonucleotide primer are respectively 5 '-cag ggc cat ttg gtt cat gt-3 ' (SEQ ID NO:1) and 5 '-tcc act cca cct cct gaa ttc t-3 ' (SEQ ID NO:2), and employed oligonucleotide probe is 5 '-aat gaa gac cat tgg ctg agc cga ga-3 ' (SEQ ID NO:3).
According to a preferred embodiment of the invention, the wherein said sample clinical sample that is suspicious JEV virus infection person.
According to a preferred embodiment of the invention, wherein said JEV virus is selected from any known varient of JEV virus.
According to a preferred embodiment of the invention, wherein said target polynucleotide is from JEV virus.
According to a preferred embodiment of the invention, wherein said nucleic acid amplification system is a polymerase chain reaction, and the circulation of said amplified reaction is approximately to repeat 35 times polymerase chain reaction circulation.
According to a preferred embodiment of the invention, wherein said nucleic acid amplification system comprise (a) hot resistant DNA polymerase, (b) 2 '-deoxynucleoside triphosphate, (c) can with the forward primer of article one chain combination of double-stranded target polynucleotide and (d) can with the reverse primer of the second chain combination of double-stranded target polynucleotide.
According to a preferred embodiment of the invention, the real-time detection architecture of wherein said fluorescence comprise can combine with target polynucleotide and two ends respectively bonded the oligonucleotide probe of fluorescence generation group and fluorescent quenching group is arranged.
According to a preferred embodiment of the invention, said method further comprises: required threshold cycle index when (1) determines that fluorescence that the target polynucleotide in the sample is produced reaches fixed threshold more than the baseline after increasing; (2) the threshold cycle index of target polynucleotide in the fixed sample is compared with the threshold cycle index of known quantity target polynucleotide in the standardized solution, to calculate the amount of target polynucleotide in the sample.
Another object of the present invention provides a kind of test kit that uses JEV virus in the real-time fluorescence quantitative PCR technology detection by quantitative clinical sample, and this test kit comprises: pure water (the DEPC H that (1) is equipped with concentrated solution, RNA extracting solution A, RNA extracting solution B, reverse transcriptase reaction system respectively, is handled through burnt ethylene carbonate
2O), the packing box of these reagent bottles of packing or pipe is separated and concentrated in pcr amplification reaction liquid I, pcr amplification reaction liquid II, diluent, negative control sample, strong positive standard substance and RNA positive reference substance and a plurality of reagent bottles that seal or pipe and (2).
According to another preferred embodiment of the present invention, the forward and the reverse primer that wherein are used for the target polynucleotide amplification system are respectively 5 '-cag ggc cat ttg gtt cat gt-3 ' (SEQ ID NO:1) and 5 '-tcc act cca cct cct gaa ttc t-3 ' (SEQ ID NO:2).
According to another preferred embodiment of the present invention, the oligonucleotide that wherein is used for target polynucleotide amplification and monitoring system is 5 '-aat gaa gac cat tgg ctg agc cga ga-3 ' (SEQ ID NO:3).
Description of drawings
Fig. 1 shows the typical curve under the Std curve window.At the template number is 10
2～10
8The reaction system of copy is carried out the TaqMan pcr analysis.When 100 copy numbers were 100, test sample got the Ct value about 37, promptly detected lower limit sensitivity and can arrive 100 copies.The slope of standard curve that drafting obtains is-3.41, is 43.2 in the Y-axis intercept, R
2=0.9927.
Fig. 2 shows weak three the positive sample curves of persistent erection.The Ct value of three samples is respectively 21.4,26.9,32.6; In conjunction with amplification curve is S shape, all can the decision bits positive.
Fig. 3 shows negative sample curve.The amplification curve of three samples is more straight broken line, does not have intersection point with the fluoroscopic examination threshold line, perhaps shows in the scope of Ct value between 35～40 and amplification curve does not have S shape feature.
The detailed content of invention
The present invention relates to real-time quantitative fluorescence PCR method and kit, it is poly-particularly to relate to the real-time qualitative quantitative fluorescence The application of the Zao phase laboratory diagnosis Zhong of synthase chain reaction method and kit Zai JEV virus infections thereof.
The invention provides a kind of use Real-Time Fluorescent Quantitative PCR Technique qualitative and quantitative detection clinical sample Zhong JEV disease The method that poison exists, the method comprises: (1) provides sample, the nucleic acid amplification that comprises the JEV virus gene genome nucleic acid The reaction Yu of system and fluorescence monitoring system detects mixture, and (2) are by the said target of amplified reaction cyclic amplification The indirect combination of target polynucleotide that polynucleotides, (3) are amplified said fluorescence generation group Yu, (4) are determined The fluorescence volume that fluorescence generation group produces, the fluorescence volume that (5) analysing amplified circulation produces afterwards is to determine target multinuclear Gan Existence and the relative quantity thereof of acid; Wherein but the nucleic acid amplification system comprises the antisense oligonucleotide primer of being combined Yu target polynucleotide Thing, but and fluorescent detection system comprise the oligonucleotide probe of being combined Yu target polynucleotide; Be characterised in that said The employed forward of amplified reaction Zhong and reverse oligonucleotide primer are respectively 5 '-cag ggc cat ttg gtt cat gt-3 ' (SEQ ID NO:1) and 5 '-tcc act cca cct cct gaa ttc t-3 ' (SEQ ID NO:2), and employed few nuclear The thuja acid probe is 5 '-aat gaa gac cat tgg ctg agc cga ga-3 ' (SEQ ID NO:3).
The normal PCR method of carrying out single end point determination after finishing based on amplified reaction is different, the real-time quantitative polymerization The enzyme chain reaction method can the Zai amplified reaction process Zhong at any time monitor the generation of amplified production, thereby greatly improved The true property of Zhun and the precision that quantitatively detect. As everyone knows, real-time fluorescence quantitative PCR is Zai pcr amplification Zhong, with In time, adds primer and 5 ', 3 ' end and is marked with respectively fluorescence report group (6-FAM amidite Suo base fluorescein for example 6) and the specific oligonucleotide probe of fluorescent quenching group (for example 6-Suo base tetramethylrhodamin). If probe Do not have to be combined Yu target complement sequence, it is complete that its sequence keeps, and the fluorescence signal of reporter group emission will be by quenching group Absorb, so there is not fluorescence signal to produce; And when pcr amplification reaction carried out, 5 ' of archaeal dna polymerase-3 ' was circumscribed The enzymatic activity fluorescence probe of will degrading causes the fluorescence report group to separate Yu the fluorescent quenching group, thereby fluorescence monitoring system System can receive and be recorded to fluorescence signal. DNA chain of every amplification is that fluorescence molecule of You produces, thereby realizes The accumulation of fluorescence signal is monitored in real time Zheng PCR reaction process, and can be borrowed Yu the PCR product forms Complete Synchronization The Zhu calibration curve carries out quantitative analysis to unknown target polynucleotide (template).
Similar to common real-time quantitative fluorescence PCR technology, test sample Zhong JEV viral genome of the present invention is two The method Zhong that the chain target polynucleotide exists comprises equally that (1) provides and comprises (a) sample to be checked, (b) heat-resistant dna Polymerase and (c) amplification reaction system of 2-deoxynucleoside triphosphate (dNTP), and comprise (a) can Yu The forward primer of article one complementary strand combination of double-stranded target polynucleotide to be checked, (b) can Yu double-stranded target multinuclear to be checked The reverse primer of the second complementary strand combination of thuja acid, and (c) can Yu arbitrary of double-stranded target polynucleotide Chain combination and two ends are marked with respectively oligonucleotide probe glimmering of fluorescence generation group and fluorescent quenching group The light detection architecture; (2) by the said target polynucleotide of one or many PCR amplification; (3) annealing After said fluorescence generation group be combined Yu target polynucleotide by probe and produce fluorescence signal; (4) detect also Determine the fluorescence volume that fluorescence generation group produces; (5) analyze the fluorescence that the one or many amplification cycles is sent Amount is to detect existing of target polynucleotide; Be characterised in that wherein said target polynucleotide is the JEV viral genome Polynucleotides, employed forward and reverse oligonucleotide primer are respectively 5 '-cag ggc cat ttg gtt cat gt-3 ' (SEQ ID NO:1) and 5 '-tcc act cca cct cct gaa ttc t-3 ' (SEQ ID NO:2), and employed few nuclear The thuja acid probe is 5 '-aat gaa gac cat tgg ctg agc cga ga-3 ' (SEQ ID NO:3).
As previously mentioned, all JEV Strain that may exist in order to detect clinical sample Zhong, and can truly have by Zhun Effect ground with the JEV virus infections Yu west nile virus, flavivirus, russian spring-summer encephalitis virus, dengue fever virus jaundice Poison belongs to virus and enterovirus, california antigenic group viruses, alphavirus and measles virus etc., and other are common The infection such as encephalitis correlated virus differentiate and come that standby Oligonucleolide primers and the probe of a little purposes of Zhe realized of design and Zhi is A very important sport technique segment. For this reason, we Zai uses suitable foranalysis of nucleic acids software to known JEV disease The genome nucleotide sequence of poison variant carries out homology relatively and Zhao goes out on the basis of homology segment, further makes The suitable primer-design software (for example Primer Express 2) of Yong is selected and meter has the lower nucleotide sequence that shows Oligonucleolide primers 1:5 '-cag ggc cat ttg gtt cat gt-3 ' is (SEQ ID NO:1) (20mrs), primer 2: 5 '-Tcc act cca cct cct gaa ttc t-3 ' is (SEQ ID NO:2) (22mrs); And the few nucleosides that shows sequence under having Acid probe: 5 '-aat gaa gac cat tgg ctg agc cga ga-3 ' is (SEQ ID NO:3) (26mrs). Primer institute is right The amplification section of answering is equivalent to JEV virus P3 Zhu NS5 gene (archaeal dna polymerase that RNA relies on) coded sequence Conserved region, length are 111bp. Wherein, primer 1 is complementary to virus genomic 9095-9114 position nucleotides; Primer 2 is complementary to virus genomic 9184-9205 position nucleotides. The complementary Yu 9159-9184 of probe Ze position Nucleotides. The designed a little primers of Zhe and the probe of You Yu all has the sequence that is complementary to the viral genome conserved region, and And do not have homology Yu the nucleotide sequence of other flavivirus and encephalitis correlated virus, do not comprise any normal yet The endonuclease of seeing is so the false negative and the false positive that greatly reduce even avoided JEV virus to detect improve Reliability and the Zhun exactness that detects.
Can use the DNA synthesizer to synthesize required Oligonucleolide primers, Yong molecular sieve and fast protein liquid phase layer Carry out the processing of ammonia solution after analysing method (FPLC) purifying. Same synthetic required probe sequence, after the ammonia solution is processed respectively Zai its 5 ' end mark Zuo is the 6-FAM amidite of fluorescence generation group (reporter group), and its 3 ' end mark of Zai is by work Zuo is the TAMRA of fluorescent quenching or inhibitor group in the coupling of property linking arm. With FPLC purification by chromatography fluorescence labeling Probe. Then, can use polyacrylamide gel (20%) electrophoresis and AAS under the Denaturing The primer that physical characterization is synthesized and probe.
Use conventional RNA extracting method or commercially available RNA to extract kit, from the early stage blood serum sample that derives from the experimenter, extract RNA, use then reverse transcription reaction system (the every microlitre You of Zai 250mM Tris-HCL (pH8.0), 250mM KCL, 20mM MgCL2With buffer solution that 50mM DTT Zu becomes Zhong contain 50 mMLV of unit enzymes and 1 The RNA of unit enzyme) the RNA Zhuan that extracts is changed into CDNA and with this reverse transcription product Yong Yu continue after pcr amplification.
Zai contain primer 1 and 2 (each 20pmol), dna profiling (pcr amplification target sequence), dNTP (10mM), The reaction system Zhong of hot resistant DNA polymerase (Plantinium archaeal dna polymerase), PCR buffer solution and water uses CDNA sequence to as above obtaining on the moving fluoroscopic examination thermal cycler of the Zi of ABI 7000 types or other structure types Carry out pcr amplification. Employed reaction condition is 95 ℃ of Yu sex change after 3 minutes, 94 ℃ 10 seconds, 60 ℃ 1 minute Zhong carries out 40 circulations altogether.
After reaction is finished, by the automatic analysis system analysis result of configuration on the Zhuan Zhi. This research of Zai Zhong is if follow Ring Yu (Ct) Zhi is equal to or greater than 35, and the result is namely negative; If Ct Zhi is less than 35, Ze is positive as a result. Its Zhong is take front 15 fluorescence signal Zuo of being produced of circulation of reaction as the fluorescence background signal, the default setting of fluorescence thresholding 10 times of standard deviation for the fluorescence signal of 3-15 circulation. In general, the Ct Zhi of each template is Yu this template The logarithm of initial copy number linear. Initial copy number Yue is many, and Ct Zhi Yue is little. Because the Zai amplified reaction In stage before the Ct Zhi Zhi, the enzyme of amplification system Zhong, primer, probe and dNTPs are all greatly excessive, and the p of system Zhi and ion concentration etc. are also relatively stable, so the efficient of amplified reaction is irrelevant the satisfying of a Yu template number at this moment And definite value. The Zhi You starting template number (copy number of polynucleotide sample) relevant Yu Ct Zhi and Yu sample are irrelevant The PCR system effectiveness.
Can utilize the standard items Zuo calibration curve processed of known initial copy number, wherein with the initial copy number of target polynucleotide Logarithm be abscissa, and take the Ct Zhi that as above records as Zong Zuo mark. After obtaining the Ct Zhi of unknown quantity target polynucleotide, Can learn from the calibration curve of the data creating that obtains based on parallel laboratory test the logarithm of its initial copy number, then The initial copy number that calculates this target polynucleotide (namely initially enters the Zhi number amplification phase when amplification cycles reaches Ct Zhi The time, the existing property of the Chong of Ct Zhi is fabulous, i.e. in same target polynucleotide Zai different time amplification or the different pipes of same time Zai The resulting Ct Zhi that increases Zong is constant). That is to say, in order to extrapolate based on above-mentioned amplified reaction result The amount of target polynucleotide (initial copy number), method of the present invention also further comprises: (1) determines sample Zhong's Required Yu cycle-index when the fluorescence that target polynucleotide produces after increasing reaches fixed threshold more than the baseline; (2) with the Yu cycle-index of fixed sample Zhong target polynucleotide Yu standard liquid Zhong known quantity target polynucleotide The Yu cycle-index compare, thereby calculate the amount of sample Zhong target polynucleotide.
Another object of the present invention provides a kind of kit that uses Real-Time Fluorescent Quantitative PCR Technique quantitatively to detect clinical sample Zhong JEV virus, and this kit comprises: (1) is Zhuan You concentrate, RNA extract A, RNA extract B, reverse transcriptase reaction system, pure water (the DEPC H that processes through burnt ethylene carbonate respectively2O), has this The pcr amplification reaction liquid I of the conventional ingredient that the field is known and II, dilution, negative control sample, strong positive mark Zhun product and RNA positive reference substance and a plurality of reagent bottles that seal or pipe and (2) are separated and are concentrated and pack Zhe The packing box of a little reagent bottles or pipe.
According to a preferred embodiment of the invention, wherein concentrated solution is used for concentrated liquid sample to be checked.
According to another preferred embodiment of the present invention, wherein diluent is used to dilute positive quantitative criterion product.
According to another preferred embodiment of the present invention, the forward and the reverse primer that wherein are used for the target polynucleotide amplification system are respectively 5 '-cag ggc cat ttg gtt cat gt-3 ' (SEQ ID NO.1) and 5 '-tcc act cca cct cct gaa ttc t-3 ' (SEQ ID NO:2).
According to another preferred embodiment of the present invention, the oligonucleotide that wherein is used for target polynucleotide amplification and monitoring system is 5 '-aat gaa gac cat tgg ctg agc cga ga-3 ' (SEQ ID NO:3).
Can according to as described above with test kit in the method pointed out in the appended working instructions use test kit of the present invention.
As previously mentioned, in order to detect all JEV virus variation bodies that may exist in the clinical sample, and can be accurately and effectively with JEV virus infection and west nile virus, yellow fever virus, fores encephalitis virus, dengue fever virus flavivirus and enterovirus, california antigenic group viruses, infection such as other common encephalitis correlated virus such as alphavirus and Measles virus are differentiated and are come, the homology that the present invention is based on these varients and correlated virus genome nucleotide sequence compares, and has designed the Oligonucleolide primers and the probe of suitable test kit of the present invention especially.The real-time quantitative that uses these primers and probe to finish detects, and has reduced false positive and false negative that JEV virus polynucleotide detect greatly.Our tolerance range that the present invention detects JEV viral genome Nucleotide that experiment showed, is almost 100%, and sensitivity reaches 10
2Individual copy/ml.
What be worth special instruction is that in order to ensure the accuracy of JEV method for detecting virus of the present invention, we have also used according to known JEV viral RNA sequence synthetic single stranded DNA as the positive criteria product.And, also use and carry the recombinant plasmid of JEV viral nucleotide (CDNA) sequence as DNA quantitative measurement standard product.By these additional criteria product, use test kit of the present invention to carry out parallel detection under the same conditions and make so-called outer marking quantitative typical curve, with the detection tolerance range and the accuracy of further checking test kit of the present invention, and as the additional quality control standard of production test kit of the present invention.
In addition, because the annealing temperature that is adopted in the amplification reaction condition of test kit of the present invention is 60 ℃, the actual annealing temperature of preset distance differs for 69 ℃ and reaches about 10 ℃.So,, also be enough to guarantee combining and the generation and the release of fluorescent signal of fluorescent probe and target nucleotide even the target nucleotide in the sample has only the variation of single Nucleotide.
Embodiment
Embodiment 1: encephalitis b virus detection kit and use thereof
(1) preparation comprises the test kit of following moiety: RNA extracting solution A (100 μ l/ pipe) 1 pipe, RNA extracting solution B (2000 μ l/ pipe) 1 pipe, PCR reaction solution I (180 μ l/ pipe) 1 pipe, reversed transcriptive enzyme system (20 μ l/ pipe) 1 pipe, PCR reaction tubes (unlabelled pipe) 10 pipes, DEPC H
2O (2000 μ l/ pipe) 1 pipe, strong positive standard substance (50 μ l/ pipe) 1 pipe, critical positive criteria product (50ul/ pipe) 1 pipe, negative control (50 μ l/ pipe) 1 pipe.
(2) collection of specimens, transport and preserve: the doubtful encephalitis B patient early stage serum specimen of falling ill is got in aseptic technique, collects in aseptic plastic test tube by the funnel-like dixie cup serum then and places the freezing preservation of cryogenic refrigerator (70 ℃ or-20 ℃).As test, sample needs to transport in environment below 0 ℃ and sent to the laboratory in 24 hours.
(3) detect step and interpretation of result:
The RNA extracting solution A that in the 0.5ml centrifuge tube that autoclaving was handled, adds the abundant mixing of 3 μ l of Xiang Yixin (RNA extracting solution A easily precipitates, and needs before the sampling inhale repeatedly with pipettor to draw after beating mixing).Add 50 μ l serum specimens or yin and yang attribute standard substance again, add the abundant mixing of RNA extracting solution B of 200 μ l then.Room temperature was placed after 5 minutes, 6000 rev/mins (rpm) centrifugal 1 minute, the careful suction removed supernatant, and wash precipitation with 75% pre-cooled ethanol (with the preparation of DEPC water) and (get 75% ethanol, 400 μ l and wash precipitation for twice, fully shake mixing, 6000 rev/mins (rpm) goes after centrifugal 1 minute to wash once with 75% ethanol, 400 μ l behind the supernatant again), 65 ℃ of dryings also precipitate 10 minutes.Pure water (the DEPC H that the diethylpyrocarbonate that must use when wherein, preparing 75% ethanol provides in the test kit is handled
2O).
Then, add 18 μ l PCR reaction solution I and reversed transcriptive enzyme is 2 μ l at sediment tube, the concussion mixing, placed 45 minutes instantaneous (3 seconds) centrifugal back 37 ℃, 95 ℃ of heating 3 minutes.After instantaneous (3 seconds) are centrifugal, draw 5 μ l reverse transcription products and carry out the PCR reaction.
With the centrifugal several seconds of strong positive standard substance 6000rpm, add diluent 901, fully be denoted as 10 behind the mixing
5Get 3 new 0.5ml centrifuge tubes then in addition, standard substance are carried out 10 times of gradient dilutions successively (be denoted as 10 respectively
4, 10
3, 10
2) ,-20 ℃ of preservations are standby.
Get sample (every part of 5ul) then respectively and with the quantitative criterion product and the negative control of volume, application of sample carries out pcr amplification in the PCR reaction system.The PCR cycling condition is: 95 ℃ of pre-sex change 3 minutes, 94 ℃ 10 seconds, 60 ℃ 1 minute, carry out 40 circulations altogether.
Reaction finishes the back and preserves the detection data file.Make the canonical plotting under typical curve (Stdcurve) window reach best (being correlation values＜-0.95) (referring to Fig. 1) according to analyzing back image adjustment analytical parameters.Can be found out respectively that by Fig. 2 and Fig. 3 the fluorescence curve of positive sample and preset threshold line have an intersection point, the fluorescence curve of negative sample then is lower than threshold value.At last the mensuration numerical value (Qty) of the not key sample that calculates by the instrument automatic analysing apparatus, the i.e. rna content of JEV virus (gene copy number/ml=8 (Qty) in the sample.
Sequence table
＜110〉China Sickness Prevention Control Center Virus Disease Prevention Control Institute
Anda gene limited-liability company of Zhongshan University
＜120〉method and the test kit of detection encephalitis b virus
<140>
<141>
<160>3
<2l0>1
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>1
cagggccatt?tggttcatgt
<210>2
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>2
tccactccac?ctcctgaatt?ct
<210>3
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, with probe as pcr amplification.
<400>3
aatgaagacc?attggctgagc?ccgaga
Claims (9)
1 one kinds are used the method that encephalitis b virus exists in the real-time fluorescence quantitative polymerase chain reaction technology detection by quantitative sample, this method comprises: (1) provides and comprises sample, nucleic acid amplification system, reaction and detection mixture with the fluorescence monitoring system, (2) by the said target polynucleotide of amplified reaction cyclic amplification, (3) said fluorescence generation group is combined with the target polynucleotide that is amplified is indirect, (4) determine the fluorescence volume that fluorescence generation group is produced, the fluorescence volume that (5) analysing amplified circulation back produces is to determine existing and relative quantity of target polynucleotide; Wherein the nucleic acid amplification system comprise can with target polynucleotide bonded Oligonucleolide primers, and the fluorescence monitoring system comprise can with target polynucleotide bonded oligonucleotide probe; Be characterised in that employed forward and reverse oligonucleotide primer are respectively 5 '-cag ggc cat ttg gtt cat gt-3 ' (SEQ ID NO:1) and 5 '-tcc act cca cct cctgaa ttc t-3 ' (SEQ ID NO:2), and employed oligonucleotide probe is 5 '-aat gaa gac cat tgg ctgagc cga ga-3 ' (SEQ ID NO:3).
2, according to the method for claim 1, wherein said nucleic acid amplification system comprise (a) hot resistant DNA polymerase, (b) 2 '-deoxynucleoside triphosphate, (c) can with the forward primer of article one chain combination of double-stranded target polynucleotide and (d) can with the reverse primer of the second chain combination of double-stranded target polynucleotide.
3, according to the process of claim 1 wherein said fluorescent detection system comprise can combine with target polynucleotide and two ends respectively bonded the oligonucleotide probe of fluorescence generation group and fluorescent quenching group is arranged.
4, according to the method for claim 1, said method further comprises: required threshold cycle index when (1) determines that fluorescence that the target polynucleotide in the sample is produced reaches fixed threshold more than the baseline after increasing; (2) the threshold cycle index of target polynucleotide in the fixed sample is compared with the threshold cycle index of known quantity target polynucleotide in the standardized solution, to calculate the amount of target polynucleotide in the sample.
5, according to the process of claim 1 wherein that said JEV virus is selected from any variant of encephalitis b virus.
6, according to the process of claim 1 wherein that said target polynucleotide is from encephalitis b virus.
7, use the test kit of encephalitis b virus in the real-time fluorescence quantitative polymerase chain reaction technology detection by quantitative clinical sample, this test kit comprises: the packing box that (1) is equipped with concentrated solution, RNA extracting solution A, RNA extracting solution B, reverse transcriptase reaction system respectively, these reagent bottles of packing or pipe are separated and concentrated in pure water, amplification reaction solution I, amplification reaction solution II, diluent, negative control sample, strong positive standard substance and the RNA positive reference substance of handling through coke acid second two fat and a plurality of reagent bottles that seal or pipe and (2).
8, according to the test kit of claim 7, the forward and the reverse primer that wherein are used for the target polynucleotide amplification system are respectively 5 '-cag ggc cat ttg gtt cat gt-3 ' and 5 '-tcc act cca cct cct gaa ttc t-3 '.
9, according to the test kit of claim 7, the oligonucleotide that wherein is used for target polynucleotide amplification and monitoring system is 5 '-aat gaa gac cat tgg ctg agc cga ga-3 '.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101781687A (en) * | 2009-01-19 | 2010-07-21 | 中国人民解放军军事医学科学院微生物流行病研究所 | Flavivirus-detecting gene chip probe and gene chip detection method |
| CN101942526A (en) * | 2010-07-05 | 2011-01-12 | 中国药品生物制品检定所 | Method for detecting real-time fluorescence quota PCR of epidemic encephalitis B virus and kit |
| CN106521033A (en) * | 2016-12-01 | 2017-03-22 | 四川出入境检验检疫局检验检疫技术中心 | Nucleic acid combination for detecting Japanese B encephalitis virus, kit and application of nucleic acid combination |
| CN106755577A (en) * | 2016-12-27 | 2017-05-31 | 湖南新南方养殖服务有限公司 | A kind of real-time fluorescence RT PCR kits for detecting japanese encephalitis virus in piglet Cord blood and application thereof |
| CN101178350B (en) * | 2006-11-09 | 2019-10-11 | 中山大学达安基因股份有限公司 | Detect the kit of rabies viruses |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101178350B (en) * | 2006-11-09 | 2019-10-11 | 中山大学达安基因股份有限公司 | Detect the kit of rabies viruses |
| CN101781687A (en) * | 2009-01-19 | 2010-07-21 | 中国人民解放军军事医学科学院微生物流行病研究所 | Flavivirus-detecting gene chip probe and gene chip detection method |
| CN101781687B (en) * | 2009-01-19 | 2012-12-12 | 中国人民解放军军事医学科学院微生物流行病研究所 | Flavivirus-detecting gene chip probe and gene chip detection method |
| CN101942526A (en) * | 2010-07-05 | 2011-01-12 | 中国药品生物制品检定所 | Method for detecting real-time fluorescence quota PCR of epidemic encephalitis B virus and kit |
| CN101942526B (en) * | 2010-07-05 | 2013-01-02 | 中国药品生物制品检定所 | Method for detecting real-time fluorescence quota PCR of epidemic encephalitis B virus and kit |
| CN106521033A (en) * | 2016-12-01 | 2017-03-22 | 四川出入境检验检疫局检验检疫技术中心 | Nucleic acid combination for detecting Japanese B encephalitis virus, kit and application of nucleic acid combination |
| CN106755577A (en) * | 2016-12-27 | 2017-05-31 | 湖南新南方养殖服务有限公司 | A kind of real-time fluorescence RT PCR kits for detecting japanese encephalitis virus in piglet Cord blood and application thereof |
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|---|---|
| CN100368557C (en) | 2008-02-13 |
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