CN101178350B - Detect the kit of rabies viruses - Google Patents
Detect the kit of rabies viruses Download PDFInfo
- Publication number
- CN101178350B CN101178350B CN200610123413.4A CN200610123413A CN101178350B CN 101178350 B CN101178350 B CN 101178350B CN 200610123413 A CN200610123413 A CN 200610123413A CN 101178350 B CN101178350 B CN 101178350B
- Authority
- CN
- China
- Prior art keywords
- pcr
- rabv
- target polynucleotide
- amplification
- fluorescence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to method and kits existing for rabies viruses (RABV) in detection clinical sample, more particularly to the method for real-time polymerase chain reaction technology early diagnosis rabies virus infection and used kit.
Description
Fields
The present invention relates to method and kits existing for rabies viruses in detection clinical sample, more particularly to fixed in real time
The rabies viruses in fluorescence polymerase chain reaction technology test sample is measured, to early diagnose the method for rabies virus infection and be made
Kit.
Background of invention
Rabies are also known as hydrophobia, are a kind of acute biography of the central nervous system of infecting both domestic animals and human caused by hydrophobin
It catches an illness, is legal category A infectious disease.Rabies are more common in the carnivores such as dog, wolf, cat.Human rabies are mainly people by illness
Caused by animal bite, or it is related with domestic animal close contact.Once morbidity, the death rate almost 100%.Clinical manifestation is distinctive mad
It is hot-tempered, frightened and restless, fear of wind is hydrophobia, salivation and pharyngismus, eventually to occur paralysis and threat to life.
Hydrophobin (Rabies virus) belongs to Rhabdoviridae (Rhabdoviridae), and viral shape is in bullet shape (60
~400nm × 60~85nm), the blunt garden in one end, other end plano-concave has cyst membrane, it is helically symmetrical to include capsid.Nucleic acid is sub-thread
Amerism strand RNA.The rabies major source of infection of developing country is disease dog, human rabies account for about 80 by sick dog disseminator~
90%, it is secondly cat and wolf.In developed country, since dog rabies are controlled, wild animal such as fox orangutan, food blood bat, skunk
The important infection sources is increasingly becoming with racoon etc..
People is generally susceptible to rabies.Rabies influence world's most area, entire U.S. state, Africa, Asia and
There are rabies in Some European area.It is reported that the whole world is every year because the number of rabies death is about ten thousand people of 4-7.Absolutely mostly
Number cases occur in developing country, wherein 98% in Asia, and China is only second to India, occupies second place of the world.It is mad in China
The major source of infection of dog disease is disease dog, and human rabies account for 80-90% by sick dog disseminator.
Rabic laboratory diagnostic method includes negri body inspection, fluorescence antibody inspection technique, Enzyme-multiplied immune technique inspection
Survey, rabies virus antigen, virus purification.
Due to rabies once falling ill, case fatality rate almost absolutely, therefore will always focus in prevention.Suspect quilt
After animal bite, being inoculated with rabies vaccine and application rabies antiserum is almost existing unique method.But due to rabies vaccine
It is expensive with rabies antiserum, so establishing a kind of more cheap, sensitive and special method of early diagnosis should have
Broad application prospect.
Viral RNA is detected using round pcr, has many advantages, such as that early detection, sensitivity and specificity are high.Real-time fluorescence is fixed
Measuring PCR technology is to be grown up based on traditional round pcr the mid-90 in last century.With traditional (end point determination) PCR
Technology is compared, and near real-time quantitative monitoring method not only realizes the quantitative analysis of very little copy number target polynucleotide, but also has
The advantages that a possibility that specificity and accuracy are stronger, the degree of automation is higher and pollutes is smaller.Therefore, the technology is more in target
In the detection and quantitative analysis of nucleotide sample, traditional PCR method has been gradually replaced.
It is well known that certain is special in using the detection of known Real-Time Fluorescent Quantitative PCR Technique and quantitative analysis clinical sample
In the practice of targeting nucleic acid, in order to reduce and avoid the false negative or false positive of testing result, the accuracy of quantitative analysis is improved,
One critical basic fundamental link be how based on known target polynucleotide sequence design and prepare primer appropriate with
Oligonucleotide probe.The present inventor is on the basis for being engaged in round pcr especially Real-Time Fluorescent Quantitative PCR Technique research for many years in the past
On, which is applied to the detection and quantitative analysis of rabies viruses genomic polynucleotide, has successfully completed the present invention.
Summary of the invention
The present invention relates to real-time quantitative fluorescence PCR method and kits, more particularly to real-time quantitative fluorescence polymerase chain
The application of reaction method and its kit in the earlier laboratory diagnosis of rabies virus infection.
Existed the present invention provides a kind of using rabies viruses in Real-Time Fluorescent Quantitative PCR Technique quantitative detection clinical sample
Method, this method comprises: (1) provide include rabies viruses genomic nucleic acids sample, nucleic acid amplification system and fluorescence monitoring
The reaction of system and detection mixture, (2) by target polynucleotide described in amplified reaction cyclic amplification, (3) make described glimmering
Light occurs group and is combined indirectly with the target polynucleotide being amplified, and (4) determine that fluorescence volume caused by group, (5) occur for fluorescence
The fluorescence volume that generates is after analysis amplification cycles to determine the presence and its relative quantity of target polynucleotide；Wherein nucleic acid amplification system packet
Include can with the Oligonucleolide primers in conjunction with target polynucleotide, and fluorescent detection system include can be with the widow in conjunction with target polynucleotide
Nucleotide probe；Forward and reverse Oligonucleolide primers used in amplified reaction described in being characterized in that are 5 '-respectively
Gacatgtttttctcccggattgagca-3 ' (SEQ ID NO:1) and 5 '-tcttcttcaaagttcttatggaa-3 ' (SEQ
ID NO:2), and used oligonucleotide probe is 5 '-gggttcataaagcagatcaatctcactgcgagag-3 '
(SEQ ID NO:3).
It is different from based on the traditional PCR method of single end point determination is carried out after the completion of amplified reaction, real-time quantitative polymerase
Chain reaction method can monitor the generation of amplified production at any time in the process of amplified reaction, to substantially increase quantitative detection
Accuracy and precision.As it is well known, real-time fluorescence quantitative PCR is while primer and 5 ', 3 ' to be added in PCR amplification
End is marked with fluorescent reporter group (such as 6-FAM amidite Fluoresceincarboxylic acid 6) and fluorescent quenching group (such as 6- respectively
Carboxyl tetramethylrhodamin) specific oligonucleotide probe.It is combined if probe is not complementary with target sequence, sequence is kept
Completely, the fluorescence signal of reporter group transmitting will be quenched group absorptions, so generating without fluorescence signal；And work as PCR amplification
When reaction carries out, 5 ' -3 ' 5 prime excision enzyme activities of archaeal dna polymerase will degrade fluorescence probe, and fluorescent reporter group is caused to be quenched with fluorescence
The group that goes out separation, thus fluorescence monitoring system can receive and fluorescence signal is recorded.Every one DNA chain of amplification has a fluorescence
Molecule generates, so that it is fully synchronized to realize that the accumulation of fluorescence signal is formed with PCR product, monitor in real time entire PCR react into
Journey, and quantitative analysis can be carried out to unknown target polynucleotide (template) by standard curve.
It is similar to common real-time quantitative fluorescence PCR technology, rabies viruses genome double-strand in test sample of the invention
Equally include (a) measuring samples (b) hot resistant DNA polymerase including (1) offer in method existing for target polynucleotide and (c) 2- is de-
The amplification reaction system of oxygen ribonucleoside triphosphote (dNTP), and including (a) can be with first of double-strand target polynucleotide to be checked
Complementary strand combine forward primer, (b) can with the reverse primer in conjunction with the Article 2 complementary strand of double-strand target polynucleotide to be checked,
And (c) can any bar chain combination with double-strand target polynucleotide and two ends be marked with respectively fluorescence occur group and
The fluorescent detection system of the oligonucleotide probe of fluorescent quenching group；(2) pass through one or many PCR amplification institutes
The target polynucleotide said；(3) make described fluorescence that group occur after annealing glimmering by the combination generation of probe and target polynucleotide
Optical signal；(4) it detects and determines that fluorescence volume caused by group occurs for fluorescence；(5) one or many amplification cycles are analyzed to be sent out
Fluorescence volume out, to detect the presence of target polynucleotide；It is characterized in that wherein described target polynucleotide is rabies viruses gene
Group polynucleotides, used forward and reverse Oligonucleolide primers are 5 '-gacatgtttttctcccggattgagca- respectively
3 ' (SEQ ID NO:1) and 5 '-tcttcttcaaagttcttatggaa-3 ' (SEQ ID NO:2), and used few core
Thuja acid probe is 5 '-gggttcataaagcagatcaatctcactgcgagag-3 ' (SEQ ID NO:3).
As previously mentioned, in order to detect all types rabies viruses that may be present street strain and vaccine in clinical sample
Strain, and can be accurately and effectively by rabies virus infection and japanese encephalitis virus, sindbis alphavirus, Colti virus, Ke Li meter
Asia-Congo hemorrhagic fever virus, clostridium tetani, Neisseria meningitidis, Coxsackie virus infection distinguish, and design and prepare reality
The Oligonucleolide primers and probe of these existing purposes are a very important sport technique segments.For this purpose, we are using appropriate
Foranalysis of nucleic acids software carries out tetraploid rice to the genome nucleotide sequence of known rabies viruses variant and finds out homologous
On the basis of section, the Oligonucleolide primers 1:5 '-with nucleotide sequence shown below is further devised
Gacatgtttttctcccggattgagca -3 ' (26mrs) (SEQ ID NO:1) and primer 2: 5 ' -
Tcttcttcaaagttcttatggaa-3 ' (23mrs) (SEQ ID NO:2)；And the oligonucleotides with sequence shown below is visited
Needle: 5 '-gggttcataaagcagatcaatctcactgcgagag -3 ' (SEQ ID NO:3).
Therefore, it is an object of the present invention to provide a kind of using in Real-Time Fluorescent Quantitative PCR Technique quantitative detection sample
The method of rabies viruses, this method comprises: (1) is provided including anti-including sample, nucleic acid amplification system and fluorescence monitoring system
Should be with detection mixture, (2) by target polynucleotide described in amplified reaction cyclic amplification, described fluorescence occurs for (3)
Group is combined indirectly with the target polynucleotide being amplified, and (4) determine that fluorescence volume caused by group occurs for fluorescence, and (5) analysis is expanded
After increasing circulation, the presence and its phase of target polynucleotide are determined according to the fluorescence volume combination quantitation curves generated in circular response
To amount (i.e. the copy number of target nucleic acid sequence)；Wherein nucleic acid amplification system include can be with the antisense oligonucleotide primer in conjunction with target polynucleotide
Object, and fluorescence real-time monitoring system includes the oligonucleotide probe that can be specifically bound with target polynucleotide；Feature exists
In used forward and reverse Oligonucleolide primers be 5 '-gacatgtttttctcccggattgagca-3 ' (SEQ ID respectively
NO:1) and 5 '-tcttcttcaaagttcttatggaa-3 ' (SEQ ID NO:2), and used oligonucleotide probe is
5 '-gggttcataaagcagatcaatctcactgcgagag-3 ' (SEQ ID NO:3).
According to a preferred embodiment of the invention, wherein described sample is the clinic of suspicious rabies virus infection person
Sample.
According to a preferred embodiment of the invention, wherein described target polynucleotide comes from rabies viruses.
According to a preferred embodiment of the invention, wherein described nucleic acid amplification system is polymerase chain reaction, and
And described amplified reaction circulation is the polymerase chain reaction circulation for about repeating 30 times.
According to a preferred embodiment of the invention, wherein described nucleic acid amplification system includes (a) heat-resistant dna polymerization
Enzyme, (b) 2 '-deoxynucleoside triphosphate, (c) can with the forward primer of first chain combination of double-strand target polynucleotide, and
It (d) can be with the reverse primer of the Article 2 chain combination of double-strand target polynucleotide.
According to a preferred embodiment of the invention, wherein described fluorescence real-time detection system includes can be more with target
Nucleotide combine and two ends respectively in connection with have fluorescence occur group and fluorescent quenching group oligonucleotide probe.
According to a preferred embodiment of the invention, described method further comprises: (1) determining that the target in sample is more
Nucleotide generated fluorescence after expanding reaches threshold cycle-index required when the fixed threshold of baseline or more；It (2) will really
The threshold cycle-index of target polynucleotide is compared with the threshold cycle-index of known quantity target polynucleotide in standard solution in fixed sample
Compared with to calculate the amount of target polynucleotide in sample.
It is a further object to provide a kind of using in Real-Time Fluorescent Quantitative PCR Technique quantitative detection clinical sample
The kit of rabies viruses, the kit include: that (1) is respectively provided with RNA extracting solution A (100 μ l/ pipe), RNA extracting solution B (4ml/
Bottle), pure water (DEPC H reverse transcriptase reaction system (20 μ l/ pipe), handled through burnt ethylene carbonate2O) (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), RABV positive qualitative reference product (1 × 106copies/ml、1×
107copies/ml、1×108copies/ml、1×109Copies/ml, each 10 μ l/ pipe) and multiple reagent bottles for sealing
Or centrifuge tube, it further include PCR reaction tube.(2) separate and concentrate the packing box for packing these reagent bottles or centrifuge tube.
Wherein RNA extracting solution A is by SiO2It is 2.0 that pH value is adjusted after DEPC water process, 4 DEG C of preservations after high pressure sterilization.RNA
Extracting solution B is by GuSCN (guanidinium isothiocyanate) 120g, 0.1M Tris-HCl (pH 6.4) 100ml 0.2M, EDTA (PH8.0)
22ml mixing composition, 4 DEG C of preservations.DEPC H2O is added DEPC dissolution in 1 ‰ ratio by pure water and mixes, and it is small to be placed at room temperature for 12
When more than.4 DEG C of preservations after high pressure sterilization.Reverse transcriptase reaction system is by mMLV proenzyme liquid (200U/ μ l), RNasin stoste
(40U/ μ l) is configured to every microlitre containing 50U mMLV enzyme, 4U RNasin, -20 DEG C of preservations with enzyme dilution buffer liquid.
Every person-portion RABV-PCR amplification reaction solution I contain each 0.05 μ l of primer PRABV1 and PRABV2 (40pmol/ μ l),
0.4 μ l of 25mM dNTPs, 5 × RT buffer, 4 μ l composition, 18 μ l of total amount, -20 DEG C of preservations.Each PCR reaction tube is by RABV
40 μ l of PCR reaction solution, 30 μ l of solid capping agent, Taq enzyme are 3 μ l composition.RABV PCR reaction solution, solid capping agent, Taq enzyme
System's composition is as follows: RABV PCR reaction solution is by primer PRABV1, each 0.25 μ l of PRABV2 (40pmol/ μ l), fluorescence probe FP
(30pmol/ μ l) 0.1 μ l, 39.4 μ l of PCR reaction buffer, -20 DEG C of preservations.Wherein PCR reaction buffer is by 50mM Tris-
HCl(pH8.0)、5mM MgCl2, 50mM KCl, 3% formamide composition, 4 DEG C preservation.
Solid capping agent is dissolved by solid slice paraffin through 70 DEG C, qualitative filter paper filtering and the analysis handled through high pressure sterilization
Neat liquid paraffin is formulated in 1: 6 ratio.
Taq enzyme system is to dilute Taq enzyme stoste (10U/ μ l), 25mM dNTPs, 3% agarose with enzyme dilution buffer liquid
At 1U/ μ l Taq enzyme, 3mM dNTPs, 0.4% agarose.- 20 DEG C of preservations.
Another preferred embodiment according to the present invention, wherein for the positive and reverse of target polynucleotide amplification system
Primer is 5 '-gacatgtttttctcccggattgagca-3 ' (SEQ ID NO:1) and 5 '-respectively
Tcttcttcaaagttcttatggaa -3 ' (SEQ ID NO:2).
Another preferred embodiment according to the present invention, wherein the few core for target polynucleotide amplification and monitoring system
Thuja acid is 5 '-gggttcataaagcagatcaatctcactgcgagag-3 (SEQ ID NO:3).
Amplification section corresponding to primer is equivalent to the conserved region of Rables virus glycoprotein gene coded sequence, length 173bp.
Wherein, primer 1 is complementary to virus genomic 701-726 nucleotide；Primer 2 is complementary to virus genomic 851-
873 nucleotide.Probe is then complementary with 800-833 nucleotide.
These primer and probes as designed by the present invention all have the sequence for being complementary to viral genome conserved region, and
And there is no homology with the nucleotide sequence of other types virus, it does not include any common endonuclease, so significantly yet
The false negative and false positive for even avoiding Rabies Virus Detection are reduced, the reliability and accuracy of detection are improved.
Oligonucleolide primers needed for the synthesis of DNA synthesizer can be used, it is laggard with molecular sieve and FPLC purification by chromatography
The processing of row ammonolysis.As fluorescence group occurs for probe sequence needed for same synthesis in its 5 ' end label respectively after ammonolysis processing
The 6-FAM amidite of (reporter group), and be coupled by active linking arm and in its 3 ' end label as fluorescent quenching or suppression
The TAMRA of group processed.With the probe of FPLC purification by chromatography fluorescent marker.Then, the polyacrylamide under Denaturing can be used
Primer and probe synthesized by amine gel (20%) electrophoresis and spectrophotometry physical characterization.
Using conventional RNA extraction method or commercially available RNA extracts kit, extracted from the saliva sample for deriving from subject
Then RNA uses reverse transcription reaction system, extracted RNA is converted to cDNA and this reverse transcription product is used for after
PCR amplification.
Containing primer 1 and 2 (each 10pmol), fluorescence probe FP (3pmol), DNA profiling (PCR amplification target sequence),
DNTP (3mM), hot resistant DNA polymerase (Taqman), PCR buffer and water reaction system in, using PE5700 type or other
PCR amplification is carried out to the cDNA sequence as above obtained on the automatic fluorescence detection thermal cycler of structure type.Used reaction
Condition was 93 DEG C of initial denaturations after 2 minutes, presses 93 DEG C first, 15 seconds → 60 DEG C, completes within 60 seconds 10 circulations, then press 93 DEG C again,
15 seconds → 60 DEG C, 60 seconds conditions carry out 30 circulations altogether.
After the reaction was completed, result is analyzed by the automatic analysis system configured on device.To 5700 (ABI of PE GeneAmp
Prism 7300, ABI Prism 7000, DA-7600, Line Gene, iCycler are referring to the method) for, if increased bent
Line is not S-type or circulation threshold (Ct) value is equal to 30, is as a result feminine gender；If growth curve is S-type and Ct value is less than 30, as a result
It is then the positive.It is as a result feminine gender if the not S-type curve of growth curve or Ct value are blank for Light Cycle；
If the S-type curve of growth curve and CT value is less than 40, as a result if for the positive.Wherein caused by preceding 15 circulations with reaction
For fluorescence signal as autofluorescent background signal, the default setting of fluorescence thresholding is the standard deviation of the fluorescence signal of 3-15 circulation
10 times.It is, in general, that the Ct value of each template and the logarithm of the starting copy number of the template are in a linear relationship.Starting copy number is got over
More, Ct value is smaller.Because of the stage before the Ct value of amplified reaction, the enzyme, primer, probe and dNTPs in amplification system are equal
It is significantly excessive, and the p value of system and ion concentration etc. are also relatively stable, so the efficiency of amplified reaction is one and mould at this time
The unrelated saturation definite value of plate number.Relevant to Ct value only initial profiling number (copy number of polynucleotide sample) and and sample
Unrelated PCR system efficiency.
Standard curve is made using the standard items of known starting copy number, wherein with target polynucleotide starting copy number
Logarithm is abscissa, and the Ct value as above to measure is ordinate.It, can be from base after the Ct value for obtaining unknown quantity target polynucleotide
In the logarithm for learning its starting copy number on the standard curve for the data creating that parallel laboratory test obtains, the target multicore is then calculated
(when amplification cycles, which reach Ct value, initially enters the exponential amplification phase, the reproducibility of Ct value is fabulous, i.e., for the starting copy number of thuja acid
Same target polynucleotide is expanded in different time or that obtained Ct value is expanded in different pipes is always constant the same time).
That is, in order to extrapolate the amount (starting copy number) of target polynucleotide based on above-mentioned amplified reaction result, it is of the invention
Method may further comprise: that (1) determines that the generated fluorescence after expanding of the target polynucleotide in sample reaches baseline or more
Required threshold cycle-index when fixed threshold；(2) the threshold cycle-index of target polynucleotide in fixed sample and standard is molten
The threshold cycle-index of known quantity target polynucleotide compares in liquid, to calculate the amount of target polynucleotide in sample.
It is a further object to provide a kind of using in Real-Time Fluorescent Quantitative PCR Technique quantitative detection clinical sample
The kit of rabies viruses, the kit include: that (1) is respectively provided with RNA extracting solution A (100 μ l/ pipe), RNA extracting solution B (4ml/
Bottle), pure water (DEPC H reverse transcriptase reaction system (20 μ l/ pipe), handled through burnt ethylene carbonate2O) (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), RABV positive qualitative reference product (1 × 106copies/ml、1×
107copies/ml、1×108copies/ml、1×109Copies/ml, each 10 μ l/ pipe) and multiple reagent bottles for sealing
Or centrifuge tube, it further include PCR reaction tube.(2) separate and concentrate the packing box for packing these reagent bottles or centrifuge tube.
According to a preferred embodiment of the invention, wherein concentrate is used for concentrated liquid measuring samples.
Another preferred embodiment according to the present invention, wherein dilution is for diluting positive plasmid standards for quantitation.
Another preferred embodiment according to the present invention, wherein for the positive and reverse of target polynucleotide amplification system
Primer is 5 '-gacatgtttttctcccggattgagca-3 ' (SEQ ID NO:1) and 5 '-respectively
Tcttcttcaaagttcttatggaa -3 ' (SEQ ID NO:2).
Another preferred embodiment according to the present invention, wherein the few core for target polynucleotide amplification and monitoring system
Thuja acid is 5 '-gggttcataaagcagatcaatctcactgcgagag-3 (SEQ ID NO:3) '.
Examination of the invention can be used according to the method pointed out in appended operation instructions as described above and in kit
Agent box.
As previously mentioned, in order to detect all rabies viruses variants that may be present in clinical sample, and can be accurate
Effectively by rabies virus infection and japanese encephalitis virus, sindbis alphavirus, Colti virus, Crimean-Congo hemorrhagic fever
The infection of other common virus such as virus, clostridium tetani, Neisseria meningitidis, Coxsackie virus distinguishes, the present invention is based on
To the tetraploid rice of these variants and associated viral gene group nucleotide sequence, it is specifically designed applicable kit of the present invention
Oligonucleolide primers and probe.The Real_time quantitative detection completed using these primer and probes, substantially reduces rabies viruses
The false positive and false negative of polynucleotides detection.Ours it is demonstrated experimentally that detect the accuracy of rabies viruses genome nucleotide
Almost 100%, sensitivity reaches 1 × 105A copy/ml.
It is worth illustrating, in order to ensure the accuracy of Rabies Virus Detection method of the invention, we take in use
Recombinant plasmid with rabies viruses nucleotide (CDNA) sequence is as DNA quantitative detection reference material.By these additional references
Product are carried out Parallel testing under the same conditions using kit of the invention and make so-called outer marking quantitative standard curve, with
The further detection accuracy and accuracy of verifying kit of the present invention, and as the additional quality for producing kit of the present invention
Control standard.
In addition, the annealing temperature as employed in the amplification reaction condition of kit of the present invention is 60 DEG C, preset distance
66.2 DEG C of practical annealing temperature difference up to 6.2 DEG C.So even if the target nucleotide in sample only has the change of single nucleotide acid
It is different, also it is enough to ensure that the generation and release of fluorescence probe and combination and fluorescence signal on target nucleotide.
Detailed description of the invention
When viral copy number is 105When, test sample obtains Ct value in 25 or so, i.e. Monitoring lower-cut sensitivity reachable 105It copies
Shellfish (Figure 1A).Figure 1B shows the standard curve under Std curve window.It is 10 for template number5~109The reaction system of copy
Carry out TaqMan PCR analysis.Drawing obtained slope of standard curve is -3.08, R=-0.997432 (Figure 1B).
Fig. 2 shows the curve of weak three positive samples in strong.The Ct value of three samples is 15.36,18.35 respectively,
23.10；It is S-shaped in conjunction with amplification curve, three can determine the position positive.
Fig. 3 shows ' negative ' specimens curve.The amplification curve of three samples is more straight broken line, with fluorescence detection threshold value
Line does not have intersection point, or display Ct value is 30 and amplification curve does not have S-shaped feature.
Fig. 4 shows 5 positive clinical sample curves, and CT value is respectively less than 30, and amplification curve is S-shaped, it can be determined that sun
Property.
Specific embodiment
(1) preparation includes the kit of following constituent: RNA extracting solution A (100 μ l/ pipe), RNA extracting solution B (4ml/
Bottle), reverse transcription enzyme system (20 μ l/1 pipe), DEPC H2O (2.0ml/ pipe), PCR reaction solution 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 qualitative reference product (1 ×
106copies/ml、1×107copies/ml、1×108copies/ml、1×109Copies/ml, each 10 μ l/ pipe).
(2) collection of specimens, transport and preservation: subject's saliva is acquired using sterile chamber, is then transferred to sterile cryopreservation tube.
It such as tests, sample need to save in -20 DEG C or less low temperature (such as -70 DEG C), liquid nitrogen or in the PBS containing 50% glycerol.
(3) detection and result:
First processing sample, positive quality control product and negative quality-control product.8000rpm is centrifuged the several seconds.Take high pressure sterilization processed
1.5ml centrifuge tube, the RNA extracting solution A of 10 μ l is added to centrifuge tube, adds sample, positive quality control product or negative Quality Control
Product add the RNA extracting solution B of 200 μ l and are uniformly mixed.It is placed at room temperature for 8000rpm after five minutes to be centrifuged 1 minute, carefully suck
Supernatant.The RNA extracting solution B of 200 μ l is added and is uniformly mixed, 8000rpm is centrifuged 1 minute, carefully sucks supernatant.With 75% it is pre-
Cold ethyl alcohol washes precipitating, and 8000rpm is centrifuged 1 minute, removes supernatant.It washes altogether twice.Then 65 DEG C, 10 minutes it is drying precipitated.To drying
Sediment tube afterwards is added 18 μ l PCR reaction solution I and beats, and adds 2 μ l of reverse transcriptase, mixes.65 DEG C are placed 45 minutes, 95 DEG C
Heating 3 minutes.8000rpm centrifugation 1 minute, for use.The RNA reverse transcription in precipitating will be adsorbed on into cDNA.After reverse transcription reaction,
It draws 5 μ l reverse transcription products and does PCR reaction.It is spare after the positive qualitative reference product centrifugation several seconds.
Then the qualitative reference product and quality-control product for taking sample (every part of 5 μ l) and same volume respectively, are loaded into PCR reaction system
In and carry out PCR amplification.PCR cycle condition is:
Then 93 DEG C → 2 minutes initial denaturations press 93 DEG C 15 seconds → 60 DEG C 60 seconds, after 10 circulations, by 93 DEG C 15 seconds → 60
DEG C 60 seconds carry out 30 circulations.
Detection data file is saved after reaction.Make standard curve (Std according to image adjustment analysis parameter after analysis
Curve) canonical plotting under window reaches best (i.e. correlation numerical value is between -1.0~-0.97) (referring to fig. 4).
It can be seen that by Fig. 4, the fluorescence curve of positive sample and the threshold line of setting have an intersection point, and the fluorescence curve of ' negative ' specimens is low
In threshold value.Last instrument automatically analyzes the unknown sample numerical value (Qty) calculated.The rabies viruses rna content of sample (copy by gene
Shellfish number/ml)=2.07 × 107Gene copy/ml (Qty).
Sequence table
Claims (1)
1. using the kit of rabies viruses target polynucleotide in Real-Time Fluorescent Quantitative PCR Technique quantitative detection clinical sample,
It is characterized in that, which includes:
(1) A, 4ml/ bottles of RNA extracting solution of RNA extracting solution B of 100 μ l/ pipes, the reverse transcriptase reaction of 20 μ l/ pipes are respectively provided with
System, the pure water of 2.0ml/ pipe handled through burnt ethylene carbonate, the RABV-PCR amplification reaction solution I of 180 μ l/ pipes, 250 μ l/
The critical positive quality control product of RABV and each 10 of the negative quality-control product of pipe, the RABV strong positive quality-control product of 200 μ l/ pipes, 200 μ l/ pipes
The 1 × 10 of μ l/ pipe6copies/ml、1×107copies/ml、1×108Copies/ml and 1 × 109Copies/ml's
RABV positive qualitative reference product, and the multiple reagent bottles or centrifuge tube sealed, further include PCR reaction tube；
(2) separate and concentrate the packing box for packing these reagent bottles or centrifuge tube；
Wherein, RNA extracting solution A is by SiO2It is 2.0 that pH value is adjusted after DEPC water process, and 4 DEG C of preservations, RNA are extracted after high pressure sterilization
Liquid B is made of the 0.2M EDTA22ml mixing of 0.1M Tris-HCl100ml, pH8.0 of guanidinium isothiocyanate 120g, pH6.4, and 4
DEG C save, the pure water handled through burnt ethylene carbonate by pure water in 1 ‰ ratio be added DEPC dissolution mix, it is small to be placed at room temperature for 12
When more than, 4 DEG C of preservations after high pressure sterilization, reverse transcriptase reaction system is former by 200U/ μ l mMLV proenzyme liquid, 40U/ μ l RNasin
Liquid is configured to every microlitre containing 50U mMLV enzyme, 4U RNasin, -20 DEG C of preservations, every person-portion RABV-PCR with enzyme dilution buffer liquid
Amplification reaction solution I contains each 0.05 μ l of 40pmol/ μ l primer PRABV1 and 40pmol/ μ l PRABV2,0.4 μ of 25mM dNTPs
L, 5 × RT buffer, 4 μ l is formed, 18 μ l of total amount, -20 DEG C of preservations；
Wherein, forward and reverse primer used in target polynucleotide amplification is 5 '-respectively
Gacatgtttttctcccggattgagca-3 ' and 5 '-tcttcttcaaagttcttatggaa-3 ', and it is wherein more for target
Amplification oligonucleotide and the oligonucleotide probe of monitoring are 5 '-gggttcataaagcagatcaatctcactgcgagag-3 '.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200610123413.4A CN101178350B (en) | 2006-11-09 | 2006-11-09 | Detect the kit of rabies viruses |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200610123413.4A CN101178350B (en) | 2006-11-09 | 2006-11-09 | Detect the kit of rabies viruses |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101178350A CN101178350A (en) | 2008-05-14 |
| CN101178350B true CN101178350B (en) | 2019-10-11 |
Family
ID=39404688
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200610123413.4A Active CN101178350B (en) | 2006-11-09 | 2006-11-09 | Detect the kit of rabies viruses |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101178350B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643929A (en) * | 2012-04-05 | 2012-08-22 | 中华人民共和国大榭出入境检验检疫局 | Consensus-degenerate hybridoligonucleotide primer (CODEHOP) reverse transcription-polymerase chain reaction (RT-PCR) reagent and method for detecting lyssavirus viruses |
| CN107016258B (en) * | 2016-01-27 | 2020-06-05 | 应清界 | Method for fluorescence quantitative calculation based on recombinase-mediated isothermal nucleic acid amplification (RAA) method |
| CN107034309B (en) * | 2016-02-03 | 2020-06-16 | 中国农业科学院兰州兽医研究所 | Real-time fluorescent RPA kit and test strip RPA kit for rapidly detecting porcine pseudorabies virus and application thereof |
| CN107868820B (en) * | 2017-10-26 | 2021-02-12 | 深圳深知生物科技有限公司 | Primer group and kit for canine multiple genetic disease screening |
| CN109439803A (en) * | 2018-12-20 | 2019-03-08 | 湖北金雀医学检验实验室有限公司 | Rabies viruses fluorescence PCR detection reagent kit |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1786190A (en) * | 2004-12-10 | 2006-06-14 | 中国疾病预防控制中心病毒病预防控制所 | Method of detecting cerebritis B virus and kit |
-
2006
- 2006-11-09 CN CN200610123413.4A patent/CN101178350B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1786190A (en) * | 2004-12-10 | 2006-06-14 | 中国疾病预防控制中心病毒病预防控制所 | Method of detecting cerebritis B virus and kit |
Non-Patent Citations (2)
| Title |
|---|
| 周斌，苏鑫铭，张素芳，贾赟，曹瑞兵，陈溥言.PCR快速检测伪狂犬病病毒野毒感染.中国病毒学19 6.2004,19(6),612-615. * |
| 张强，唐青，刘卫滨，李浩，梁国栋.狂犬病毒TaqManPCR检测方法的建立.中华流行病学杂志27 10.2006,27(10),889-893. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101178350A (en) | 2008-05-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110551846B (en) | Cpf1 kit for quickly detecting African swine fever virus nucleic acid and detection method thereof | |
| CN111004870B (en) | Novel coronavirus N gene nucleic acid detection kit | |
| CN106947838B (en) | African swine fever virus non-structural gene real-time fluorescence LAMP (loop-mediated isothermal amplification) detection primer group, kit and detection method | |
| CN106957927B (en) | African swine fever fluorescent PCR detection reagent, African swine fever fluorescent PCR detection kit and application thereof | |
| CN107513584B (en) | A kind of five heavy fluorescence quantitative kits detecting enterovirus | |
| CN110273027B (en) | Nucleic acid typing detection kit and detection method for norovirus GII, GII and GIV | |
| CN101886138A (en) | Three-color fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) combined detection method of enterovirus 71, Coxsackie virus A16 and other subtypes of enterovirus as well as kit thereof | |
| CN110699489B (en) | Real-time fluorescence PCR detection primer probe set, kit and method for African swine fever virus CD2V gene | |
| CN101178350B (en) | Detect the kit of rabies viruses | |
| CN105603123A (en) | Real-time fluorescence RPA reagent kit and test strip RPA reagent kit for rapidly detecting porcine parvovirus and application of reagent kits | |
| CN101144771B (en) | Reagent kit for detecting human HIV | |
| CN105648114B (en) | Fluorescent RT-PCR (reverse transcription-polymerase chain reaction) primer, probe and kit for detecting new variant type highly pathogenic porcine reproductive and respiratory syndrome virus and detection method | |
| CN112739833A (en) | Primer pair, probe and kit for detecting SARS-CoV-2 by utilizing nested RPA technology and application thereof | |
| CN105002298B (en) | A kind of fluorescent quantitative PCR detection method of huichun viremia virus | |
| CN107083446B (en) | Diarrhea pathogenic bacteria multiple gene detection system and kit and application thereof | |
| CN114085929B (en) | Kit for detecting African swine fever virus wild strain and vaccine strain | |
| CN113186312B (en) | Molecular marker for distinguishing Brucella A19 vaccine strain and wild strain | |
| CN104774953A (en) | Fluorescent PCR (polymerase chain reaction) detection reagent for African swine fever virus (ASFV) CP530R gene, and preparation method and application thereof | |
| CN112226539A (en) | Norovirus nucleic acid detection kit | |
| Qian et al. | Clustered regularly interspaced short palindromic Repeat/Cas12a mediated multiplexable and portable detection platform for GII genotype Porcine Epidemic Diarrhoea Virus Rapid diagnosis | |
| CN112111609A (en) | Universal nucleic acid detection kit for enteroviruses | |
| CN109321683A (en) | A kind of A type Sai Nika viral diagnosis primer, kit, method for detecting virus and application | |
| CN109628640B (en) | RPA-LFD primer, method and kit for rapidly detecting spring viremia of carp virus | |
| CN110157836B (en) | Primer, probe and method for detecting IBRV and BVDV | |
| CN100368557C (en) | Method of detecting cerebritis B virus and kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Open date: 20080514 |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP03 | Change of name, title or address | ||
| CP03 | Change of name, title or address |
Address after: 510665 No. 19 incense Hill Road, hi tech Industrial Development Zone, Guangdong, Guangzhou Patentee after: Guangzhou Da'an gene Co.,Ltd. Patentee after: INSTITUTE FOR VIRAL DISEASE CONTROL AND PREVENTION, CHINESE CENTER FOR DISEASE CONTROL AND PREVENTION Address before: 510665, No. 19, Xiangshan Road, Guangzhou hi tech Industrial Development Zone, Guangzhou, Guangdong, Guangdong Patentee before: DA AN GENE CO., LTD. OF SUN YAT-SEN University Patentee before: INSTITUTE FOR VIRAL DISEASE CONTROL AND PREVENTION, CHINESE CENTER FOR DISEASE CONTROL AND PREVENTION |