CN100424183C - Method and its kit for early testing SARS virus infection - Google Patents
Method and its kit for early testing SARS virus infection Download PDFInfo
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Abstract
The present invention relates to a method and a reagent kit for detecting the existence of severe acute respiratory syndrome pathogens namely SARS viruses, particularly to a method and a reagent kit for early diagnosing SARS virus infection by a real-time quantitative fluorescence polymerase chain reaction technology.
Description
Affiliated field
The present invention relates to detect the pathogenic agent of SARS (Severe Acute Respiratory Syndrome) in the clinical sample--method and test kit that SARS virus exists particularly relate to the method and the employed test kit that infect with real-time quantitative fluorescence polymerase chain reaction technique early diagnosis SARS virus.
Background of invention
SARS (Severe Acute Respiratory Syndrome) (SARS) is the lethality transmissible disease that causes because of any novel varient that infects coronavirus.At first find in Chinese Guangdong province some areas for the end of the year 2002, and promptly propagate into 25 countries and regions, the world and other many provinces and cities of China in 3 months very soon, accumulative total reported cases number reaches 8202 people, wherein 725 people's death.This viral infectivity is strong, the serious harm people's health and lives.Patient's onset is anxious, and antibiotic therapy is often invalid.Spreading rate height in the crowd causes epidemic situation outburst and mortality ratio higher easily.
At present, the SARS virus laboratory detection technology of The World Health Organization's approval mainly contains three kinds: the one, and enzyme-linked immunosorbent assay (ELISA), this method can detect the existence of antibody in the serum reliably, but need finding that symptom just can obtain reliable detected result after 21 days, therefore be difficult to early diagnosis.The 2nd, immunofluorescence technique (IFA), this method generally just can detect antibody in back 10 days in infection, and needed fixedly SARS virus, and must use immunofluorescence microscopy to finish by veteran technician.More than two kinds of methods all need measure and relatively acute phase and convalescent serum antibody titer, time span is long, is unfavorable for patient's early diagnosis and therapy.The third is reverse transcriptional PCR method (RT-PCR), and this method falls ill by detecting patient that the RNA fragment of SARS virus realizes early diagnosis in respiratory secretions in back 10 days or the movement equal samples.The RT-PCR method is that at present all are applied to the most early stage, convenient and special method in the several different methods of SARS Infect And Diagnose, and it is saved patient's life and ward off disease to propagate and played great role early stage quick diagnosis SARS.
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 very little 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.Therefore, this technology replaces traditional PCR method gradually in the detection and quantitative analysis of target polynucleotide sample, obtains very using widely.
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 SARS 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 SARS 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 '-gac gag ttc gtg gtg gtg ac-3 ' (SEQ IDNO:1) and 5 '-aag ctt ctg ggc cag ttc ct-3 ' (SEQ ID NO:2), and employed oligonucleotide probe is 5 '-caa aat gaa aga gct cag ccc cag atg-3 ' (SEQ IDNO:3).
According to a preferred embodiment of the invention, the wherein said sample clinical sample that is suspicious SARS virus the infected.
According to a preferred embodiment of the invention, wherein said SARS virus is selected from any known varient of SARS virus.
According to a preferred embodiment of the invention, wherein said target polynucleotide is from SARS 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 SARS 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 a preferred embodiment of the invention, wherein concentrated solution is used for concentrated liquid sample to be checked (virion of sample is attached together and rapid precipitation).
According to another preferred embodiment of the present invention, wherein diluent is used to dilute quantitative positive criteria 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 '-gac gag ttc gtg gtg gtg ac-3 ' (SEQ ID NO:1) and 5 '-aagctt ctg ggc cag ttc ct-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 '-caa aat gaa aga gct cag ccc cag atg-3 (SEQ ID NO:3) '.
Description of drawings
Fig. 1 shows the typical curve under the Std curve window
Fig. 2 shows weak three the positive sample curves of persistent erection
Fig. 3 shows negative sample curve
The detailed content of invention
The present invention relates to real-time quantitative fluorescence PCR method and kit, particularly relate to the real-time quantitative fluorescence polymerization The application in the early stage laboratory diagnosis that SARS virus infects of enzyme chain reaction method and kit thereof.
The invention provides a kind of use Real-Time Fluorescent Quantitative PCR Technique and quantitatively detect SARS disease in the clinical sample The method that poison exists, the method comprises: (1) provides sample, the nucleic acid that comprises the SARS virus genomic nucleic acids The reaction of amplification system and fluorescence monitoring system and detection mixture, (2) are by amplified reaction cyclic amplification institute The target polynucleotide of saying, (3) make said fluorescence generation group and the indirect combination of target polynucleotide that is amplified, (4) determine the fluorescence volume that fluorescence generation group produces, the fluorescence volume that (5) analysing amplified circulation produces afterwards with Determine existence and the relative quantity thereof of target polynucleotide; Wherein the nucleic acid amplification system comprises and can be combined with target polynucleotide Oligonucleolide primers, and fluorescent detection system comprises the oligonucleotide probe that can be combined with target polynucleotide; Be characterised in that employed forward and reverse oligonucleotide primer are respectively 5 '-gac in the said amplified reaction Gag ttc gtg gtg gtg ac-3 ' (SEQ ID NO:1) and 5 '-aag ctt ctg ggc cag ttc Ct-3 ' (SEQ ID NO:2), and employed oligonucleotide probe is 5 '-caa aat gaa aga Gct cag ccc cag atg-3 ' (SEQ ID NO:3).
From finish based on amplified reaction after to carry out the normal PCR method of single end point determination different, real-time quantitative is poly-The synthase chain reaction method can be monitored the generation of amplified production at any time in the process of amplified reaction, thereby greatly carries High accuracy and the precision of quantitative detection. As everyone knows, real-time fluorescence quantitative PCR is at pcr amplification In, add simultaneously primer and 5 ', 3 ' end and be marked with respectively fluorescence report group (6-FAM amidite for example Fluoresceincarboxylic acid 6) and the specific oligonucleotide of fluorescent quenching group (for example 6-carboxyl tetramethylrhodamin) Probe. If probe is not combined with target complement sequence, it is complete that its sequence keeps, the fluorescence of reporter group emission Signal will be absorbed by quenching group, so there is not fluorescence signal to produce; And when pcr amplification reaction carries out, DNA 5 ' of polymerase-3 ' the 5 prime excision enzyme activity fluorescence probe of will degrading causes fluorescence report group and fluorescent quenching base Group separates, thereby the fluorescence monitoring system can receive and be recorded to fluorescence signal. DNA chain of every amplification namely has one Individual fluorescence molecule produces, thereby realizes that the accumulation of fluorescence signal and PCR product form Complete Synchronization, in real time prison Survey whole PCR reaction process, and can quantitatively divide unknown target polynucleotide (template) by calibration curve Analyse.
Similar to common real-time quantitative fluorescence PCR technology, SARS virus gene in the test sample of the present invention Organize and comprise equally in the method that double-stranded target polynucleotide exists that (1) provides and comprise that (a) sample to be checked (b) is anti-Hot archaeal dna polymerase and (c) 2 '-amplification reaction system of deoxynucleoside triphosphate (dNTP), and comprise (a) The forward primer that can be combined with article one complementary strand of double-stranded target polynucleotide to be checked (b) can be with to be checked two The reverse primer of the second complementary strand combination of chain target polynucleotide, and (c) can with double-stranded target multinuclear glycosides Arbitrary chain combination of acid and two ends are marked with respectively widow's nuclear of fluorescence generation group and fluorescent quenching group The fluorescent detection system of thuja acid probe; (2) by the said target multinuclear of one or many PCR amplification Thuja acid; (3) said fluorescence generation group is combined by probe with target polynucleotide and produces fluorescence Signal; (4) detect and fluorescence volume that definite fluorescence generation group produces; (5) analyze the one or many amplification The fluorescence volume that circulation is sent is to detect existing of target polynucleotide; Be characterised in that wherein said target multinuclear glycosides Acid is SARS virus genome polynucleotides, and employed forward and reverse oligonucleotide primer are respectively 5 '-gac gag ttc gtg gtg gtg ac-3 ' (SEQ ID NO:1) and 5 '-aag ctt ctg ggc Cag ttc ct-3 ' (SEQ ID NO:2), and employed oligonucleotide probe is 5 '-caa aat Gaa aga gct cag ccc cag atg-3 ' (SEQ ID NO:3).
As previously mentioned, in order to detect all SARS virus variants that may exist in the clinical sample, and energy Enough accurately and effectively with SARS virus infection and influenza A virus, Respiratory Syncytial Virus(RSV), parainfluenza virus Distinguish Deng other common respiratory virus infections, design and preparation realize these purposes Oligonucleolide primers and Probe is a very important sport technique segment. For this reason, we are using suitable foranalysis of nucleic acids software to known The genome nucleotide sequence of all 16 SARS virus variants carry out homology relatively and find out homologous region On the basis of section, further use suitable primer-design software (for example Primer Express 3) to have Show down the Oligonucleolide primers 1:5 ' of nucleotide sequence-gac gag ttc gtg gtg gtg ac-3 ' (20 Mrs) (SEQ ID NO:1) and primer 2: 5 '-aag ctt ctg ggc cag ttc ct-3 ' (20 Mrs) (SEQ ID NO:2); And the oligonucleotide probe that shows sequence under having: 5 '-caa aat gaa aga Gct cag ccc cag atg-3 ' (SEQ ID NO:3). The corresponding amplification section of primer is equivalent to SARS The conserved region of the RNA polymerase coded sequence of virus variant TOR2, length are 85bp. Wherein, primer 1 Be complementary to virus genomic 28382-28400 position nucleotides; Primer 2 is complementary to virus genomic The nucleoside agents box acid of 28447-28466 position. Probe is complementary and 28404-28432 position nucleotides then. Because institute The design these primers and probe all have the sequence that is complementary to the viral genome conserved region, and with other breathings The nucleotide sequence of road virus does not have homology, does not comprise any common endonuclease, so greatly subtract yet Less even false negative and the false positive of having avoided SARS virus to detect, the reliability and the degree of accuracy that detect have been improved.
Can use the DNA synthesizer to synthesize required Oligonucleolide primers, pure with molecular sieve and FPLC chromatography Carrying out the ammonia solution after the change processes. Same synthetic required probe sequence, after the ammonia solution is processed respectively at its 5 ' end mark As the 6-FAM amidite of fluorescence generation group (reporter group), and its 3 ' end mark by activity Linking arm coupling and as the TAMRA of fluorescent quenching or inhibitor group. With FPLC purification by chromatography fluorescence labeling Probe. Then, can use polyacrylamide gel (20%) electrophoresis and spectrophotometric under the Denaturing Primer and probe that the method physical characterization is synthesized.
Use conventional RNA extracting method or commercially available RNA to extract kit, from the rinse liquid sample that derives from the experimenter, extract RNA, use then the reverse transcription reaction system (at every microlitre by 250mM Tris-HCL (pH8.0), 250mM KCL, 20mM MgCL2With contain 50 mMLV of unit enzymes and 1 RNA of unit enzyme in the buffer solution of 50mM DTT) RNA that extracts is changed intocDNA and with this reverse transcription product be used for continue after pcr amplification.
In the reaction system that contains primer 1 and 2 (each 25pmol), dna profiling (pcr amplification target sequence), dNTP (10mM), hot resistant DNA polymerase (Taqman), PCR buffer solution and water, use on the automatic fluoroscopic examination thermal cycler of ABI 5700 types or other structure types as above obtainingcDna sequence dna carries out PCR and expands Increase. Employed reaction condition is 93 ℃ of denaturations after 2 minutes, at first by 93 ℃, and 15 seconds → 55 ℃, 15 Second → 72 ℃, finished 10 circulations in 20 seconds, and then by 93 ℃, 15 seconds → 58 ℃, 30 seconds conditions are altogether Carry out 35 circulations.
After reaction is finished, by the automatic analysis system analysis result of the upper configuration of device. In this research, if Circulation threshold (Ct) value is equal to or greater than 35, and the result is namely negative; If the Ct value is less than 35, the result then is sun The property. Wherein with front 15 fluorescence signals of being produced of circulation of reaction as the fluorescence background signal, fluorescence thresholding Default setting be 10 times of standard deviation of the fluorescence signal of 3-15 circulation. In general, each template Ct value and the logarithm of the initial copy number of this template linear. Initial copy number is more many, and the Ct value is more little. Because the stage before the Ct of amplified reaction value, the enzyme in the amplification system, primer, probe and dNTPs are equal Greatly excessive, and the p value of system and ion concentration etc. are also relatively stable, and this moment, the efficient of amplified reaction was one The individual saturated definite value that has nothing to do with the template number. Relevant with the Ct value only have starting template number (polynucleotide sample Copy number) and with the irrelevant PCR system effectiveness of sample.
Can utilize the standard items production standard curve of known initial copy number, wherein with the initial copy of target polynucleotide The logarithms of number are abscissa, take the Ct value that as above records as ordinate. Obtain the Ct of unknown quantity target polynucleotide After the value, can learn the right of its initial copy number from the calibration curve of the data creating that obtains based on parallel laboratory test Number, the initial copy number that calculates then this target polynucleotide (namely initially enters when amplification cycles reaches the Ct value The index amplification is during the phase, and the reappearance of Ct value is fabulous, and namely same target polynucleotide is in different time amplification or same The time resulting Ct value that increases in the difference pipe is always constant). That is to say, for based on above-mentioned expansion Increase reaction result and extrapolate the amount of target polynucleotide (initial copy number), method of the present invention also further comprises: (1) determines that fluorescence that the target polynucleotide in the sample produces reaches the fixed threshold more than the baseline after increasing The threshold cycle-index of Shi Suoxu; (2) threshold cycle-index and the standard of target polynucleotide in the fixed sample is molten The threshold cycle-index of known quantity target polynucleotide is compared in the liquid, thereby calculates target polynucleotide in the sample Amount.
Another object of the present invention provides a kind of kit that uses Real-Time Fluorescent Quantitative PCR Technique quantitatively to detect SARS virus in the clinical sample, and this kit comprises: pure water (the DEPC H that (1) is equipped with respectively concentrate, RNA extract A, RNA extract B, reverse transcriptase reaction system, is processed through burnt ethylene carbonate2O), PCR Amplification reaction solution I, pcr amplification reaction liquid II, dilution, negative control sample, strong positive standard items and RNA positive reference substance and a plurality of reagent bottles that seal or pipe and (2) are separated and are concentrated and pack these examinations The packing box of agent bottle or pipe.
According to a preferred embodiment of the invention, wherein concentrate is used for concentrated liquid sample to be checked.
According to another preferred embodiment of the present invention, wherein dilution is used for diluting positive plasmid standards for quantitation.
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 '-gac gag ttc gtg gtg gtg ac-3 ' (SEQ ID NO:1) and 5 ,-aagctt ctg ggc cag ttc ct-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 '-caa aat gaa aga gct cag ccc cag atg-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 16 SARS virus varients that may exist in the clinical sample, and can be accurately and effectively other common respiratory virus infections such as SARS virus infection and influenza A virus, respiratory syncytial virus, parainfluenza virus be distinguished, 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 the SARS virus polynucleotide detect greatly.Our tolerance range that detects the SARS virus genome nucleotide that experiment showed, is almost 100%, and sensitivity reaches 8 * 10
2Individual copy/ml.
What be worth special instruction is, accuracy in order to ensure SARS virus detection method of the present invention, we have also used according to known SARS virus RNA sequence synthetic, and the RNA sequence (108bp) of extending with export-oriented two ends at pcr amplification zone (83bp) is as RNA positive criteria product (A and B).And, also use carry SARS virus Nucleotide (
cDNA) recombinant plasmid of sequence is 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 58 ℃, the actual annealing temperature of preset distance differs for 66.2 ℃ and reaches 8.2 ℃.So,, also be enough to guarantee fluorescent probe and combining and the generation and the release of fluorescent signal on target nucleotide even the target nucleotide in the sample has only the variation of single Nucleotide.
Embodiment
(1) preparation comprises the test kit of following moiety: concentrated solution (5ml/1 bottle), RNA extracting solution A (100 μ l/ pipe), RNA extracting solution B (4ml/ bottle), reversed transcriptive enzyme system (20 μ l/1 pipe), DEPC H
2O (2.0ml/ pipe), PCR reaction solution I (180 μ l/ pipe), PCR reaction solution II (10 pipe), diluent (0.5ml/ pipe), negative control sample (50 μ l/ pipe), strong positive standard substance (2 * 10
5Gene copy/μ l; 10 μ l/ pipe), RNA positive reference substance.
(2) collection of specimens, transport and preserve: use sample solution (about 5ml stroke-physiological saline solution) fully to gargle, collect in aseptic plastic test tube by the funnel-like dixie cup rinse liquid then and place the freezing preservation of cryogenic refrigerator (70 ℃).As test, sample needs to transport in environment below 0 ℃ and sent to the laboratory in 24 hours.
(3) detection and result:
At first handle sample, RNA positive reference substance and negative control standard substance.Every part of RNA positive reference substance, sample and negative control product are joined new in the 1.5ml centrifuge tube that autoclaving was handled respectively.Add 500 μ l concentrated solutions to centrifuge tube ,-20 ℃ leave standstill 10 minutes after, 4 ℃ 12000 is centrifugal 10 minutes.After centrifugal, supernatant discarded also stays raffinate 20～30ul.The RNA extracting solution A that adds 10 μ l in residual liquid adds the RNA extracting solution B of 400 μ l again and mixes.Room temperature was placed after 5 minutes low-speed centrifugal 1 minute, and careful the suction removed supernatant, washes precipitation with 75% pre-cooled ethanol, washes altogether twice.Then 65 ℃ drying precipitated.
Then, the RNA reverse transcription that will be adsorbed on the precipitation in the presence of reversed transcriptive enzyme (2 μ l) becomes c-DNA.Behind the reverse transcription reaction, draw 5 μ l reverse transcription products and do the PCR reaction.
With the centrifugal several seconds of strong positive standard substance 6000rpm, add diluent 90 μ l, fully be denoted as 10 behind the mixing
5Get 3 new 0.5ml centrifuge tubes of sterilization 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, the application of sample performing PCR amplification of advancing to go forward side by side in the PCR reaction system.The PCR cycling condition is:
Pre-sex change in 93 ℃ → 2 minutes, then by 93 ℃ 15 seconds → 55 ℃ 15 seconds → 72 ℃ 20 seconds, do 10 circulations earlier, at last by 93 ℃ 15 seconds → 58 ℃ 30 seconds, do 35 circulations.
Reaction finishes the back and preserves the detection data file.Make the canonical plotting under typical curve (Std curve) window reach best (being dependency correlation numerical value＜-0.95) (referring to Fig. 1) according to analyzing back image adjustment analytical parameters.Can find out respectively that by Fig. 2 and Fig. 3 the fluorescence curve of positive sample and preset threshold line have an intersection point, and the fluorescence curve of negative sample is lower than threshold value.The unknown sample numerical value (Qty) that last instrument automatic analyser is calculated.The SARS virus rna content of sample (gene copy number/ml)=8 * (Qty).
Sequence table
＜110〉Da
＜120〉method and the test kit of the infection of early detection SARS virus
<140>
<141>
<160>3
<210>1
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>1
gacgagttcg?tggtggtgac
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>2
aagcttctgg?gccagttcct
<210>3
<211>27
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, with probe as pcr amplification.
<400>3
caaaatgaaa?gagctcagcc?ccagatg
Claims (3)
1. one kind is used the method that SARS virus exists in the real-time fluorescence quantitative polymerase chain reaction technology detection by quantitative sample, this method comprises: (1) provides and comprises sample, comprise hot resistant DNA polymerase, 2 '-deoxynucleoside triphosphate, can with the forward primer of article one chain combination of double-stranded target polynucleotide and can with the nucleic acid amplification system of the reverse primer of the second chain combination of double-stranded target polynucleotide, and the reaction of fluorescence monitoring system and detection 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, 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 wherein employed forward and reverse oligonucleotide primer are respectively 5 '-gac gag ttc gtg gtg gtg ac-3 ' and 5 '-aag ctt ctgggc cag ttc ct-3 ' in the nucleic acid amplification system, and wherein in the fluorescence monitoring system employed oligonucleotide probe be 5 '-caa aatgaa aga gct cag ccc cag atg-3 '.
According to the process of claim 1 wherein said fluorescence monitoring system comprise can combine with target polynucleotide and two ends respectively bonded the oligonucleotide probe 5 '-caa aat gaa aga gct cag ccc cag atg-3 ' of fluorescence generation group and fluorescent quenching group is arranged.
3. use the test kit of SARS virus in the real-time fluorescence quantitative PCR technology detection by quantitative clinical sample, this test kit comprises: (1) is equipped with concentrated solution respectively, RNA extracting solution A, RNA extracting solution B, the reverse transcriptase reaction system, pure water through the processing of coke acid second two fat, pcr amplification reaction liquid I, pcr amplification reaction liquid II, diluent, the negative control sample, strong positive standard substance and RNA positive reference substance and a plurality of reagent bottles or the pipe that seal, (2) packing box of separation and concentrated these reagent bottles of packing or pipe, be characterised in that the forward and the reverse primer that use in the target polynucleotide amplification are respectively 5 '-gac gag ttc gtg gtg gtg ac-3 ' and 5 '-aag ctt ctg ggc cag ttc ct-3 ', and wherein be used for the target polynucleotide amplification and the oligonucleotide probe of monitoring is 5 '-caa aat gaa aga gct cag ccc cag atg-3 '.
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| CN1238456A (en) * | 1999-02-12 | 1999-12-15 | 中山医科大学科技开发公司 | PCR method for testing nucleic acid by fluoremtetry and its reagent box |
| US6617162B2 (en) * | 2001-12-18 | 2003-09-09 | Isis Pharmaceuticals, Inc. | Antisense modulation of estrogen receptor alpha expression |
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