CN101178350A - Method and reagent box for testing rabies viruses - Google Patents

Abstract

The invention relates to a method for the detection of the existence of rabies virus (RABV) in the clinical sample and a kit, in particular to the method for using a real-time quantitative fluorescence polymerase chain reaction technology for the early diagnosis of the rabies virus infection and the kit which is used.

Description

Detect the method and the kit of rabies viruses

Affiliated field

The present invention relates to detect rabies viruses exists in the clinical sample method and kit, particularly relate to the rabies viruses in the real-time quantitative fluorescence polymerase chain reaction technique test sample, with the method and the employed kit of early diagnosis rabies virus infection.

Background of invention

Rabies are called hydrophobia again, and the central nervous system acute infectious disease of a kind of infecting both domestic animals and human that causes for hydrophobin is legal category A infectious disease.Rabies are more common in carnivores such as dog, wolf, cat.Human rabies mainly is due to the people is bitten by infected animal, or contact is relevant closely with domestic animal.In case morbidity, mortality ratio almost 100%.Clinical manifestation be distinctive manic, frightened and restless, be afraid of that wind fears water, hydrostomia and pharyngismus, the threat to life to paralysis takes place eventually.

Hydrophobin (Rabies virus) belongs to Rhabdoviridae (Rhabdoviridae), and viral profile is the bullet shape, and (other end plano-concave has cyst membrane for 60～400nm * 60～85nm), the blunt garden of an end, includes capsid symmetry in the shape of a spiral.Nucleic acid is sub-thread amerism strand RNA.The main infection sources of the rabies of developing country is the disease dog, and human rabies accounts for 80～90% by sick dog blazer, is cat and wolf secondly.In developed country, because dog rabies Be Controlled, wild animal such as fox orangutan, food blood bat, skunk and racoon etc. become the important infection sources gradually.

The people is to the general susceptible of rabies.Rabies have influence on most of area, the world, and all there are rabies whole U.S. state, Africa, Asia and European some areas.It is reported that whole world every year, the number because of rabies death was about 4-7 ten thousand people.Most cases occur in developing country, and wherein 98% in the Asia, and China is only second to India, occupies the second place of the world.In China, the rabic main infection sources is the disease dog, and human rabies accounts for 80-90% by sick dog blazer.

Rabic laboratory diagnostic method comprises negri body inspection, fluorescence antibody inspection technique, Enzyme-multiplied immune technique detection, rabies virus antigen, virus separation.

Because rabies are morbidity in a single day, therefore case fatality rate almost absolutely always will focus in the prevention.After suspection was bitten by animal, inoculation rabies vaccine and application rabies antiserum almost were existing unique methods.But because rabies vaccine and rabies antiserum cost an arm and a leg, so set up a kind of more cheap, sensitivity and special method of early diagnosis should be with a wide range of applications.

Utilize round pcr to detect viral RNA, have early detection, sensitivity and specificity advantages of higher.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 test of very little copy number target polynucleotide, but also has specificity and advantage such as degree of accuracy is stronger, automaticity is higher and contamination of heavy is littler.Therefore, this technology replaces traditional PCR method gradually in the detection and quantitative test of target polynucleotide sample.

As everyone knows, in using known real-time fluorescence quantitative PCR technology for detection and quantitative test 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 testing result, improve the accuracy of quantitative test, 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 research in the past for many years, and this technology is applied to the detection and the quantitative test of rabies viruses genome polynucleotide, has successfully finished the present invention.

Summary of the invention

The present invention relates to real-time quantitative fluorescence PCR method and kit, particularly relate to the application in the early stage laboratory diagnosis of rabies virus infection of real-time quantitative fluorescence polymerase chain reaction method and kit thereof.

The invention provides a kind of method that rabies viruses exists in the real-time fluorescence quantitative PCR technology detection by quantitative clinical sample of using, this method comprises: (1) provides the sample that comprises the rabies viruses genomic nucleic acids, the nucleic acid amplification system, reaction and detection potpourri 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 comprises the Oligonucleolide primers that can combine with target polynucleotide, and fluorescent detection system comprises the oligonucleotide probe that can combine with target polynucleotide; Be characterised in that employed forward and reverse oligonucleotide primer are respectively 5 '-gacatgtttttctcccggattgagca-3 ' (SEQ ID NO:1) and 5 '-tcttcttcaaagttcttatggaa-3 ' (SEQID NO:2) in the said amplified reaction, and employed oligonucleotide probe is 5 '-gggttcataaagcagatcaatctcactgcgagag-3 ' (SEQID NO:3).

With finish based on amplified reaction after to carry out the conventional P CR method of single end point determination different, the real-time quantitative polymerase chain reaction method can be monitored the generation of amplified production at any time in the process of amplified reaction, thereby has improved the accuracy and the precision of detection by quantitative greatly.As everyone knows, real-time fluorescence quantitative PCR is in pcr amplification, adds the specific oligonucleotide probe that primer and 5 ', 3 ' end are marked with fluorescence report group (for example 6-FAM amidite Fluoresceincarboxylic acid 6) and fluorescent quenching group (for example 6-carboxyl tetramethylrhodamin) respectively simultaneously.If probe does not combine with target complement sequence, its sequence is kept perfectly, and the reporter group fluorescent signal emitted will be absorbed by quenching group, so there is not fluorescence signal to produce; And when pcr amplification reaction carried out, 5 ' of archaeal dna polymerase-3 ' the 5 prime excision enzyme activity fluorescence probe of will degrading cause the fluorescence report group to separate with the fluorescent quenching group, thereby the fluorescence monitoring system can receive and record fluorescence signal.DNA chain of every amplification promptly has a fluorescence molecule to produce, thereby realizes that the accumulation of fluorescence signal and PCR product form fully synchronously, monitor whole PCR reaction process in real time, and can carry out quantitative test to unknown target polynucleotide (template) by typical curve.

Similar to common real-time quantitative fluorescence PCR technology, comprising equally in the method that the double-stranded target polynucleotide of rabies viruses genome exists in the test sample of the present invention that (1) provides comprises (a) sample to be checked (b) hot resistant DNA polymerase and (c) amplification reaction system of 2-deoxynucleoside triphosphate (dNTP), and comprise the forward primer that (a) can combine with article one complementary strand of double-stranded target polynucleotide to be checked, (b) reverse primer that can combine, and the fluorescent detection system that (c) can be marked with the oligonucleotide probe of fluorescence generation group and fluorescent quenching group respectively with arbitrary the chain combination and two ends of double-stranded target polynucleotide with the second complementary strand of double-stranded target polynucleotide to be checked; (2) by the said target polynucleotide of one or many PCR amplification; (3) make said fluorescence generation group produce fluorescence signal with combining of target polynucleotide after the annealing by probe; (4) detect and fluorescence volume that definite fluorescence generation group is produced; (5) analyze the fluorescence volume that the one or many amplification cycles is sent, to detect existing of target polynucleotide; Be characterised in that wherein said target polynucleotide is rabies viruses genome polynucleotide, employed forward and reverse oligonucleotide primer are respectively 5 '-gacatgtttttctcccggattgagca 3 ' (SEQ ID NO:1) and 5 '-tcttcttcaaagttcttatggaa-3 ' (SEQ ID NO:2), and employed oligonucleotide probe is 5 '-gggttcataaagcagatcaatctcactgcgagag-3 ' (SEQ ID NO:3).

As previously mentioned, in order to detect all types rabies viruses street strain and the vaccine strain that may exist in the clinical sample, and can accurately and effectively rabies virus infection and japanese encephalitis virus, sindbis alphavirus, Colti virus, crimean-Congo hemorrhagic fever virus, clostridium tetani, Neisseria meningitidis, Coxsackie virus infection be distinguished, design and preparation realize that the Oligonucleolide primers and the probe of these purposes are very important sport technique segments.For this reason, we carry out homology relatively and find out on the basis of homology segment at the genome nucleotide sequence that uses suitable foranalysis of nucleic acids software to known rabies viruses variant, have further designed to have Oligonucleolide primers 1:5 '-gacatgtttttctcccggattgagca-3 ' (26mrs) (SEQ ID NO:1) and primer 2: the 5 '-tcttcttcaaagttcttatggaa-3 ' (23mrs) (SEQID NO:2) that shows nucleotide sequence down; And the oligonucleotide probe that shows sequence under having: 5 '-gggttcataaagcagatcaatctcactgcgagag-3 ' (SEQ ID NO:3).

Therefore, an object of the present invention is to provide a kind of method of using rabies viruses 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 potpourri, (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 circular response; Wherein the nucleic acid amplification system comprises the Oligonucleolide primers that can combine with target polynucleotide, and the fluorescence real-time monitoring system comprises an oligonucleotide probe that can combine with the target polynucleotide specificity; Be characterised in that employed forward and reverse oligonucleotide primer are respectively 5 ' gacatgtttttctcccggattgagca-3 ' (SEQ ID NO:1) and 5 '-tcttcttcaaagttcttatggaa-3 ' (SEQ ID NO:2), and employed oligonucleotide probe is 5 '-gggttcataaagcagatcaatctcactgcgagag-3 ' (SEQ ID NO:3).

According to a preferred embodiment of the invention, the wherein said sample clinical sample that is suspicious rabies virus infection person.

According to a preferred embodiment of the invention, wherein said target polynucleotide is from rabies viruses.

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 30 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 comprises and can combine with target polynucleotide and the two ends oligonucleotide probe that fluorescence generation group and fluorescent quenching group are arranged of combination respectively.

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 standard solution, to calculate the amount of target polynucleotide in the sample.

Another object of the present invention provides a kind of kit that uses rabies viruses in the real-time fluorescence quantitative PCR technology detection by quantitative clinical sample, and this kit comprises: pure water (the DEPC H that (1) is equipped with RNA extract A (100 μ l/ pipe), RNA extract B (4ml/ bottle), reverse transcriptase reaction system (20 μ l/ pipe) respectively, is handled through burnt ethylene carbonate ₂O) (2.0ml/ pipe), RABV-PCR amplification reaction solution I (180 μ l/ pipe), negative quality-control product (250 μ l/ pipe), RABV strong positive quality-control product (200 μ l/ pipe) and the critical positive quality control product of RABV (200 μ l/ pipe), the positive quantitatively reference material (1 * 10 of RABV ⁶Copies/ml, 1 * 107copies/ml, 1 * 10 ⁸Copies/ml, 1 * 10 ⁹Copies/ml, each 10 μ l/ pipe) and a plurality of reagent bottles or the centrifuge tube that seal, also comprise the PCR reaction tube.(2) packing box of separation and concentrated these reagent bottles of packing or centrifuge tube.

Wherein RNA extract A is by SiO ₂Regulating pH value after the DEPC water treatment is 2.0,4 ℃ of preservations behind the autoclaving.RNA extract B is by GuSCN (guanidinium isothiocyanate) 120g, 0.1M Tris-HCl (pH6.4) 100ml 0.2M, and EDTA (PH8.0) 22ml mixes composition, 4 ℃ of preservations.DEPC H ₂O adds DEPC dissolving mixing by pure water in 1 ‰ ratio, and room temperature is placed more than 12 hours.4 ℃ of preservations behind the autoclaving.The reverse transcriptase reaction system is mixed with every microlitre with enzyme dilution buffer liquid and is contained 50U mMLV enzyme, 4U RNasin ,-20 ℃ of preservations by mMLV proenzyme liquid (200U/ μ l), RNasin stoste (40U/ μ l).

Everyone part RABV-PCR amplification reaction solution I contains primer PRABV1 and PRABV2 (40pmol/ μ l) each 0.05 μ l, 25mM dNTPs 0.4 μ l, 5 * RT buffer, 4 μ l form total amount 18 μ l ,-20 ℃ of preservations.Each PCR reaction tube is that 3 μ l form by RABV PCR reactant liquor 40 μ l, solid capping agent 30 μ l, Taq enzyme.RABV PCR reactant liquor, solid capping agent, Taq enzyme are composed as follows: RABV PCR reactant liquor is by primer PRABV1, PRABV2 (40pmol/ μ l) each 0.25 μ l, fluorescence probe FP (30pmol/ μ l) 0.1 μ l, PCR reaction buffer 39.4 μ l ,-20 ℃ of preservations.Wherein the PCR reaction buffer is by 50mM Tris-HCl (pH8.0), 5mM MgCl ₂, 50mM KCl, 3% formamide form 4 ℃ of preservations.

Through 70 ℃ of dissolvings, qualitative filter paper filters with the analysis neat liquid paraffin of handling through autoclaving formulated in 1: 6 ratio the solid capping agent by solid slice paraffin.

Taq enzyme system is with Taq proenzyme liquid (10U/ μ l), 25mM dNTPs, 3% agarose, is diluted to 1U/ μ l Taq enzyme, 3mM dNTPs, 0.4% agarose with enzyme dilution buffer liquid.-20 ℃ of preservations.

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 '-gacatgtttttctcccggattgagca-3 ' (SEQ ID NO:1) and 5 '-tcttcttcaaagttcttatggaa-3 ' (SEQ ID NO:2).

According to another preferred embodiment of the present invention, the oligonucleotides that wherein is used for target polynucleotide amplification and monitoring system is 5 '-gggttcataaagcagatcaatctcactgcgagag-3 (SEQ ID NO:3).

The pairing amplification section of primer is equivalent to the conserved region of rabies viruses nucleoprotein coded sequence, and length is 173bp.Wherein, primer 1 is complementary to virus genomic 701-726 position nucleotide; Primer 2 is complementary to virus genomic 851-873 position nucleotide.The then complementary and 800-833 position nucleotide of probe.

Because designed these primers and the probe of the present invention all has the sequence that is complementary to the viral genome conserved region, and there is not homology with the nucleotide sequence of other types virus, do not comprise any common endonuclease yet, so the false negative and the false positive that significantly reduce even avoided rabies viruses to detect have improved the reliability and the accuracy that detect.

Can use the DNA synthesizer to synthesize required Oligonucleolide primers, separate processing with carrying out ammonia behind molecular sieve and the FPLC purification by chromatography.Same synthetic required probe sequence, ammonia are separated after the processing respectively at the 6-FAM amidite of its 5 ' end mark as fluorescence generation group (reporter group), and at its 3 ' end mark by active linking arm coupling and as fluorescent quenching or suppress the TAMRA of group.With the fluorescently-labeled probe of FPLC purification by chromatography.Then, the primer and the probe that can use polyacrylamide gel (20%) electrophoresis under the sex change condition and spectrophotometric method physical characterization to be synthesized.

Use conventional RNA extracting method or commercially available RNA to extract kit, from the saliva sample that derives from the experimenter, extract RNA, use the reverse transcription reaction system then, with the RNA that is extracted change into CDNA and with this reverse transcription product be used to continue after pcr amplification.

In the reaction system that contains primer 1 and 2 (each 10pmol), fluorescence probe FP (3pmol), dna profiling (pcr amplification target sequence), dNTP (3mM), hot resistant DNA polymerase (Taqman), PCR damping fluid and water, on the automatic fluoroscopic examination thermal cycler of use PE5700 type or other structure types the CDNA sequence that as above obtains is carried out pcr amplification.Employed reaction conditions is 93 ℃ of pre-sex change after 2 minutes, at first by 93 ℃, 15 seconds → 60 ℃, finished 10 circulations in 60 seconds, and then by 93 ℃, 15 seconds → 60 ℃, 60 seconds conditions is carried out 30 times altogether and circulated.

After reaction is finished, go up the automatic analysis system analysis result of configuration by device.For PE GeneAmp 5700 (ABI Prism7300, ABI Prism 7000, DA-7600, Line Gene, iCycler are with reference to the method), if growth curve is not S-type or circulation threshold (Ct) value equals 30, the result is promptly negative; If growth curve is S-type and the Ct value less than 30, the result is then positive.For Light Cycle, if not S-type curve of growth curve or Ct value are blank, the result is promptly negative; If S-type curve of growth curve and CT value are less than 40, the result is then positive.Wherein with preceding 15 fluorescence signals of being produced of circulation of reaction as the fluorescence background signal, the default setting of fluorescence thresholding is 10 times of standard deviation of 3-15 round-robin fluorescence signal.In general, the logarithm of the initial copy number of the Ct value of each template and this template is linear.Initial copy number is many more, and the Ct value is more little.Because the stage before the Ct of amplified reaction value, enzyme in the amplification system, primer, probe and dNTPs are all excessive greatly, and the p value of system and ion concentration etc. are also relatively stable, so the efficient of amplified reaction is a saturated definite value that has nothing to do with the template number at this moment.The PCR system effectiveness having only starting template number (copy number of polynucleotide sample) and with sample have nothing to do relevant with the Ct value.

Can utilize the standard items production standard curve of known initial copy number, wherein the logarithm with the initial copy number of target polynucleotide is a horizontal ordinate, and is ordinate with the Ct value that as above records.After obtaining the Ct value of unknown quantity target polynucleotide, can learn the logarithm of its initial copy number from the typical curve of the data creating that obtains based on parallel laboratory test, the initial copy number that calculates this target polynucleotide then (promptly initially enters the index amplification during phase when amplification cycles reaches the Ct value, the reappearance of Ct value is fabulous, and promptly same target polynucleotide is always constant in different time amplification or the same time resulting Ct value that increases in the difference pipe).That is to say, in order to extrapolate the amount (initial copy number) of target polynucleotide based on above-mentioned amplified reaction result, method of the present invention also 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 standard solution, thereby calculate the amount of target polynucleotide in the sample.

Another object of the present invention provides a kind of kit that uses rabies viruses in the real-time fluorescence quantitative PCR technology detection by quantitative clinical sample, and this kit comprises: pure water (the DEPC H that (1) is equipped with RNA extract A (100 μ l/ pipe), RNA extract B (4ml/ bottle), reverse transcriptase reaction system (20 μ l/ pipe) respectively, is handled through burnt ethylene carbonate ₂O) (2.0ml/ pipe), RABV-PCR amplification reaction solution I (180 μ l/ pipe), negative quality-control product (250 μ l/ pipe), RABV strong positive quality-control product (200 μ l/ pipe) and the critical positive quality control product of RABV (200 μ l/ pipe), the positive quantitatively reference material (1 * 10 of RABV ⁶Copies/ml, 1 * 10 ⁷Copies/ml, 1 * 10 ⁸Copies/ml, 1 * 10 ⁹Copies/ml, each 10 μ l/ pipe) and a plurality of reagent bottles or the centrifuge tube that seal, also comprise the PCR reaction tube.(2) packing box of separation and concentrated these reagent bottles of packing or centrifuge tube.

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 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 ' gacatgtttttctcccggattgagca-3 ' (SEQ ID NO:1) and 5 '-tcttcttcaaagttcttatggaa-3 ' (SEQ ID NO:2).

According to another preferred embodiment of the present invention, the oligonucleotides that wherein is used for target polynucleotide amplification and monitoring system is 5 '-gggttcataaagcagatcaatctcactgcgagag-3 (SEQ ID NO:3) '.

Can according to as described above with kit in the method pointed out in the appended operation instructions use kit of the present invention.

As previously mentioned, in order to detect all rabies viruses variants that may exist in the clinical sample, and can be accurately and effectively other common virus such as rabies virus infection and japanese encephalitis virus, sindbis alphavirus, Colti virus, crimean-Congo hemorrhagic fever virus, clostridium tetani, Neisseria meningitidis, Coxsackie virus be infected and distinguish, the homology that the present invention is based on these variants and correlated virus genome nucleotide sequence compares, and has designed the Oligonucleolide primers and the probe of suitable 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 rabies viruses polynucleotide detect greatly.Our degree of accuracy that detects the rabies viruses genome nucleotide that experiment showed, is almost 100%, and sensitivity reaches 1 * 10 ⁵Individual copy/ml.

What be worth special instruction is that in order to ensure the accuracy of rabies viruses detection method of the present invention, we use and carry the recombinant plasmid of rabies viruses nucleotide (CDNA) sequence as DNA detection by quantitative reference material.By these additional reference product, use 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 degree of accuracy and the accuracy of further checking kit of the present invention, and as the additional quality control standard of production kit of the present invention.

In addition, because the annealing temperature that is adopted in the amplification reaction condition of kit of the present invention is 60 ℃, the actual annealing temperature of preset distance differs for 66.2 ℃ and reaches 8.2 ℃.So,, also be enough to guarantee fluorescence probe and combining and the generation and the release of fluorescence signal on target nucleotide even the target nucleotide in the sample has only the variation of single nucleotide.

Description of drawings

When viral copy number is 10 ⁵The time, test sample gets the Ct value about 25, promptly detects lower limit sensitivity and can arrive 10 ⁵Copy (Figure 1A).Figure 1B shows the typical curve under the Std curve window.At the template number is 10 ⁵～10 ⁹The reaction system of copy is carried out the TaqMan pcr analysis.The slope of standard curve that drafting obtains is-3.08, R=-0.997432 (Figure 1B).

Fig. 2 shows the curve of weak three positive samples of persistent erection.The Ct value of three samples is respectively 15.36,18.35,23.10; In conjunction with amplification curve is S shape, and the three 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 show the Ct value be 30 and amplification curve do not have S shape feature.

Fig. 4 shows 5 clinical positive sample curves, and the CT value is all less than 30, and amplification curve is a S shape, all can be judged to be the positive.

Embodiment

(1) preparation comprises the kit of following constituent: RNA extract A (100 μ l/ pipe), RNA extract B (4ml/ bottle), reverse transcriptase system (20 μ l/1 pipe), DEPC H ₂O (2.0ml/ pipe), PCR reactant liquor I (180 μ l/ pipe), negative control sample (250 μ l/ pipe), strong positive quality-control product (200 μ l/ pipe), critical positive quality control product (200 μ l/ pipe), positive quantitatively reference material (1 * 10 ⁶Copies/ml, 1 * 10 ⁷Copies/ml, 1 * 10 ⁸Copies/ml, 1 * 10 ⁹Copies/ml, each 10 μ l/ pipe).Wherein RNA extract A by---form; RNA extract B by---form; Reverse transcriptase system comprises----; PCR reactant liquor I by---form.Use among the present invention-as negative control; Use---as the strong positive quality-control product; Use and--also use----as the quantitative reference material of the positive as critical positive quality control product.

(2) collection of specimens, transport and preserve: use sterile chamber to gather person under inspection's saliva, then change aseptic frozen pipe over to.As test, sample needs in low temperature below-20 ℃ (for example-70 ℃), liquid nitrogen or contains among the PBS of 50% glycerine to preserve.

(3) detection and result:

At first handle sample, positive quality control product and negative quality-control product.The centrifugal several seconds of 8000rpm.Get the 1.5ml centrifuge tube that autoclaving was handled, add the RNA extract A of 10 μ l, add sample, positive quality control product or negative quality-control product again, add the RNA extract B of 200 μ l again and mix to centrifuge tube.Room temperature was placed after 5 minutes 8000rpm centrifugal 1 minute, and careful the suction removed supernatant.Add the RNA extract B of 200 μ l and mix, centrifugal 1 minute of 8000rpm, the careful suction removed supernatant.Pre-cooled ethanol with 75% is washed precipitation, and centrifugal 1 minute of 8000rpm removes supernatant.Wash altogether twice.65 ℃ then, 10 minutes are drying precipitated.Add 18 μ l PCR reactant liquor I to dried sediment tube and beat evenly, add reverse transcriptase 2 μ l again, mixing.Placed 45 minutes for 65 ℃, 95 ℃ were heated 3 minutes.Centrifugal 1 minute of 8000rpm, stand-by.The existence RNA reverse transcription that will be adsorbed on the precipitation down become cDNA.Behind the reverse transcription reaction, draw 5 μ l reverse transcription products and do the PCR reaction.It is positive that quantitatively reference material is standby after the centrifugal several seconds.

Get sample (every part 5 μ l) then respectively and with the quantitative reference material and the quality-control product 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 → 60 ℃ 60 seconds, after 10 circulations, carried out 30 circulations in 60 seconds by 93 ℃ 15 seconds → 60 ℃.

Reaction finishes the back and preserves the detection data file.Make the canonical plotting under typical curve (Std curve) window reach best (being that correlation numerical value is between-1.0～-0.97) (referring to Fig. 4) according to analyzing back image adjustment analytical parameters.As seen from Figure 4, 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 rabies viruses rna content of sample (gene copy number/ml)=2.07 * 10 ⁷Gene copy/ml (Qty).

Sequence table

＜110〉Da

＜120〉method and the kit of detection rabies viruses

<140>

<141>

<160>3

<210>1

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification.

<400>1

gacatgtttt?tctcccggat?tgagca

<210>2

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification.

<400>2

tcttcttcaa?agttcttatg?gaa

<210>3

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉according to the specific nucleotide sequence design, with probe as pcr amplification.

<400>3

gggttcataa?agcagatcaa?tctcactgcg?agag

Claims (6)

1. one kind is used the method that rabies viruses 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 potpourri, (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 comprises the Oligonucleolide primers that can combine with target polynucleotide, and the fluorescence monitoring system comprises the oligonucleotide probe that can combine with target polynucleotide; Be characterised in that wherein employed forward and reverse oligonucleotide primer are respectively 5 '-gacatgtttttctcccggattgagca-3 ' and 5 '-tcttcttcaaagttcttatggaa-3 ' in the nucleic acid amplification system, and wherein in the fluorescence monitoring system employed oligonucleotide probe be 5 '-gggttcataaagcagatcaatctcactgcgagag-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 5 '-gacatgtttttctcccggattgagca-3 ' of article one chain combination of double-stranded target polynucleotide and (d) can with the reverse primer 5 '-tcttcttcaaagttcttatggaa-3 ' of the second chain combination of double-stranded target polynucleotide.

3. can combine with target polynucleotide and two ends the oligonucleotide probe 5 '-gggttcataaagcagatcaatctcactgcgagag-3 ' that fluorescence generation group and fluorescent quenching group are arranged of combination respectively according to the process of claim 1 wherein that said fluorescent detection system comprises.

4. according to the process of claim 1 wherein that said method further comprises in (5) step: 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 standard solution, to calculate the amount of target polynucleotide in the sample.

5. according to the process of claim 1 wherein that said target polynucleotide is from rabies viruses.

6. use the kit of rabies viruses in the real-time fluorescence quantitative PCR technology detection by quantitative clinical sample, this kit comprises: (1) be equipped with RNA extract A (100 μ l/ pipe), RNA extract B (4ml/ bottle), reverse transcriptase reaction system (20 μ l/ pipe) respectively, through pure water (the DEPC H of burnt ethylene carbonate processing ₂O) (2.0ml/ pipe), RABV-PCR amplification reaction solution I (180 μ l/ pipe), negative quality-control product (250 μ l/ pipe), RABV strong positive quality-control product (200 μ l/ pipe) and the critical positive quality control product of RABV (200 μ l/ pipe), the positive quantitatively reference material (1 * 10 of RABV ⁶Copies/ml, 1 * 10 ⁷Copies/ml, 1 * 10 ⁸Copies/ml, 1 * 10 ⁹Copies/ml, each 10 μ l/ pipe) and a plurality of reagent bottles or the centrifuge tube that seal, also comprise the PCR reaction tube.(2) packing box of separation and concentrated these reagent bottles of packing or centrifuge tube, be characterised in that the forward and the reverse primer that use in the target polynucleotide amplification are respectively 5 '-gacatgtttttctcccggattgagca-3 ' and 5 '-tcttcttcaaagttcttatggaa-3 ', and wherein be used for the target polynucleotide amplification and the oligonucleotide probe of monitoring is 5 '-gggttcataaagcagatcaatctcactgcgagag-3 '.