CN104745724A - Primers, probe and kit used for detecting JC viruses (JCVs) - Google Patents
Primers, probe and kit used for detecting JC viruses (JCVs) Download PDFInfo
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- CN104745724A CN104745724A CN201510052911.3A CN201510052911A CN104745724A CN 104745724 A CN104745724 A CN 104745724A CN 201510052911 A CN201510052911 A CN 201510052911A CN 104745724 A CN104745724 A CN 104745724A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses primers, probe and kit used for detecting JC viruses (JCVs), belonging to the technical field of biology. The primers and probe comprise a forward primer, a reverse primer and a probe, which are used for detecting JCVs. The kit comprises the primers and the probe. The primers, the probe and the kit have the characteristic of high sensitivity and can be used for accurately detecting whether samples are infected with the JCVs.
Description
Technical field
The present invention relates to biological technical field, particularly a kind of primer, probe and test kit for detecting JC virus.
Background technology
JC virus (JC virus, JCV) belongs to papovaviridae polyomavirus kind human polyomavirus branch.Pagett in 1971 etc. isolate JC virus first from progressive multifocal leukoencephalopathy (progressive multifocalleucoencephalopathy, PML) patient's cerebral tissue.JC virus extensively exists in crowd, mainly hides in the kidney etc. of people is organized.The infection of JC virus occurs in the childhood of people, in continuing subclinical latent infection state, immunity can be asymptomatic throughout one's life under perfecting state, belong to symptomless infection, but then can cause the series of disease such as PML for AIDS patient or the patient that takes immunosuppressive drug, PML is a kind of lethality central nervous system demyelination.Recent study shows, JC virus is relevant to the generation of multiple human tumor, comprises cerebral tumor, colorectal cancer, cancer of the stomach, esophagus cancer and B cell lymphsystem tumor etc.Along with the increase year by year of China HIV/AIDS patient numbers, JC virus infection also can increase year by year as the opportunistic infection that HIV/AIDS is relevant, and the Organ Transplantation Patients such as renal transplantation is after using immunosuppressive drug, the probability that JC virus infection causes PML increases, and the harm that JC virus is brought also can be more outstanding.
The detection of JC virus infection is comprised and detects specific VP1 antibody in serum, the detection of JC viral DNA is carried out to the urine of the infected, cerebrospinal fluid, blood and pathological tissues, in situ hybridization and immunohistochemical methods detection etc. are carried out to living tissue.Because JC virus infection rate in crowd is very high, antibody test accurately can not prove that whether sample is by reactivity JC virus infection.
Summary of the invention
In order to solve the inaccurate problem of antibody test in prior art, embodiments provide a kind of primer, probe and test kit for detecting JC virus.Described technical scheme is as follows:
On the one hand, embodiments provide a kind of primer for detecting JC virus and probe, described primer and probe comprise: for detect JC virus forward primer, for detecting the reverse primer of JC virus and the probe for detecting JC virus, wherein,
The described forward primer for detecting JC virus is as shown in SEQ ID NO.1 in sequence table;
The described reverse primer for detecting JC virus is as shown in SEQ ID NO.2 in sequence table;
The described probe for detecting JC virus is as shown in SEQ ID NO.3 in sequence table;
5 ' end of described probe is connected with fluorophor FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy5, and 3 ' end of described probe is connected with quenching group TAMRA, Eclipse, BHQ1, BHQ2, BHQ3 or DABCYL.
On the other hand, embodiments provide a kind of test kit for detecting JC virus, described test kit comprises above-mentioned primer and probe.
Particularly, described test kit also comprises: nucleic acid releasing agent, PCR reaction solution, critical positive quality control product, positive quality control product, negative quality control product and working standard.
Particularly, described working standard comprises: working standard 1, working standard 2, working standard 3 and working standard 4, wherein,
Described working standard 1 is 1 × 10
7the gene fragment of the described JC virus of copy/mL;
Described working standard 2 is 1 × 10
6the gene fragment of the described JC virus of copy/mL;
Described working standard 3 is 1 × 10
5the gene fragment of the described JC virus of copy/mL;
Described working standard 4 is 1 × 10
4the gene fragment of the described JC virus of copy/mL.
Particularly, described positive quality control product is 1.0 × 10
6the T antigen gene fragment of the described JC virus of copy/mL.
Particularly, the final concentration of described forward primer and described reverse primer is 0.05-0.9 μM, and the final concentration of described probe is 0.05-0.9 μM.
Particularly, each component of described PCR reaction solution comprises in pcr amplification reaction system: dNTPs, 10 × PCR damping fluid and final concentration that the Taq enzyme that final concentration is 0.01U/ μ L ~ 0.05U/ μ L, final concentration are 0.2 ~ 0.6mM are the solution containing Mg ion of 1.5 ~ 5.0mM.
Particularly, the Triton X-100 that described nucleic acid releasing agent comprises sterilized water, final concentration is 0.03-0.3%, final concentration to be Nonyl pheno (40) ether of 0.04-0.4% and pH value be 8.3 final concentration be three (methylol) aminomethane of 0.01-0.1M.
Particularly, described critical positive quality control product is 1.0 × 10
4the gene fragment of the described JC virus of copy/mL.
Particularly, described negative quality control product is the physiological saline of concentration 0.8%.
The beneficial effect that the technical scheme that the embodiment of the present invention provides is brought is: primer and probe for detecting JC virus that the embodiment of the present invention provides, there is highly sensitive characteristic, accurately can detect sample and whether infect JC virus, the test kit for detecting JC virus that the embodiment of the present invention provides, there is highly sensitive characteristic, accurately can detect sample and whether infect JC virus, instrument is adopted to collect fluorescent signal, avoid the subjectivity that naked eyes judge, its detected result is reliable, improve the sensitivity of detection, even if this test kit of object fragment that only there is single copy also can effectively detect, detection speed is fast, and whole testing process only needs 2 ~ 3 hours altogether.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in the embodiment of the present invention, below the accompanying drawing used required in describing embodiment is briefly described, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 is the amplified fluorescence graphic representation that the embodiment of the present invention 2 provides;
Fig. 2 is the amplified fluorescence graphic representation that the embodiment of the present invention 3 provides;
Fig. 3 is the amplified fluorescence graphic representation that the embodiment of the present invention 4 provides;
Fig. 4 is the amplified fluorescence graphic representation of the typical curve that the embodiment of the present invention 5 provides;
Fig. 5 is the amplified fluorescence graphic representation that the embodiment of the present invention 5 provides;
Fig. 6 is the canonical plotting obtained by Fig. 4 that the embodiment of the present invention 5 provides, and Fig. 6 X-coordinate is LogStarting Quantity copy number, and ordinate zou is Threshold Cycle.
In figure: Cycles is cycle number, RFU is fluorescent value, and Log Starting Quantity copy number is the logarithmic value of varicella zoster virus initial concentration, and Ct (Threshold Cycle) value is cycle number; Sample 7 in sample 6,7a-embodiment 2 in sample 5,6a-embodiment 2 in sample 4,5a-embodiment 2 in sample Isosorbide-5-Nitrae a-embodiment 2 in 1a-embodiment 2.
Embodiment
For making the object, technical solutions and advantages of the present invention clearly, below in conjunction with accompanying drawing, embodiment of the present invention is described further in detail.In following examples, agents useful for same and probe are mainly purchased from precious biotechnology (Dalian) company limited.
Embodiment 1. 1 kinds is for the primer of JC viral nucleic acid detection by quantitative and fluorescent probe
Embodiments provide a kind of primer for detecting JC virus and probe, primer and probe comprise: for detect JC virus forward primer, for detecting the reverse primer of JC virus and the probe for detecting JC virus, wherein,
For detecting the forward primer of JC virus as shown in SEQ ID NO.1 in sequence table;
For detecting the reverse primer of JC virus as shown in SEQ ID NO.2 in sequence table;
For detecting the probe of JC virus as shown in SEQ ID NO.3 in sequence table;
5 ' end of probe is connected with fluorophor FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy5, and 3 ' end of probe is connected with quenching group TAMRA, Eclipse, BHQ1, BHQ2, BHQ3 or DABCYL.In embodiments of the present invention, the fluorescent reporter group of probe is the excitation wavelength of FAM, FAM is 485nm, and reception wavelength is 527nm; Quenching group is Eclipse.Way of purification can be selected from: HAP, PAGE and HPLC way of purification, and primer and probe are as following table particularly:
Table 1.JC virus primer and probe
In table 1: F:forward, forward; JC virus-F represents the forward primer of JC virus.R:reverse, oppositely; JC virus-R represents the reverse primer of JC virus.P:probe, fluorescent probe (probe); JC virus-P represents JC FLuorescent probe, and fluorescent probe can be TaqMan-MGB fluorescent probe, LNA fluorescent probe or MGB fluorescent probe.The primer of the design of table 1 and probe all entrust precious biotechnology (Dalian) company limited to synthesize.
The primer that the embodiment of the present invention provides and probe have highly sensitive characteristic, and accurately can detect the content of the JC virus in testing sample.
Embodiment 2. 1 kinds is for detecting the test kit of JC virus
Embodiments provide a kind of for detecting the quantitative test kit of JC viral nucleic acid, this test kit comprises the primer and probe that the embodiment of the present invention 1 provides, and this test kit also comprises: nucleic acid releasing agent, PCR reaction solution, critical positive quality control product, positive quality control product, negative quality control product and working standard.
Particularly, each component of PCR reaction solution comprises in pcr amplification reaction system: dNTPs, 10 × PCR damping fluid and final concentration that the Taq enzyme that final concentration is 0.01U/ μ L ~ 0.05U/ μ L, final concentration are 0.2 ~ 0.6mM are the solution containing Mg ion of 1.5 ~ 5.0mM.In the present embodiment, the proportioning of each component concentration of PCR reaction solution is: dNTPs2 μ L, 10 × PCR Buffer 5 μ L and concentration that the Taq enzyme 0.3 μ L that concentration is 5U/ μ L, concentration are 10mmol/L are the MgCl of 25mmol/L
2solution 5 μ L.
Particularly, the final concentration of forward primer and reverse primer is 0.05-0.9 μM (forward primer and reverse primer are 0.05-0.9 μM), and the final concentration of probe is 0.05-0.9 μM.In the present embodiment, concentration is that 10 μm of ol/L primers add 2.5 μ L, and concentration is that 10 μm of ol/L probes add 2.5 μ L.
In actual application, primer and probe together can be added in PCR reaction solution and form reaction system, then to add sterilized water to volume be 49.5 μ L.
Particularly, the final concentration of nucleic acid releasing agent: Triton-X100 (Triton X-100) is 0.03%, the final concentration of NP-40 (Nonyl pheno (40) ether) be 0.04% and pH value be 8.3 final concentration be the Tris-HCL (three (methylol) aminomethane) of 0.01M, solvent is sterilized water.
Particularly, negative quality control product to be concentration be 0.8% physiological saline.Weigh 0.008g solid sodium chloride and be dissolved in 1ml distilled water, mixing, directly draw 0.5 μ L and make negative quality control product.
Particularly, positive quality control product comprises containing concentration is 1.0 × 10
6the solution of the JC virogene fragment of copy/mL.Get containing JC virus liquid, strain is by China typical culture collection center (China Center for TypeCultureCollection, CCTCC) provide, after cultivating, get and add after isopyknic nucleic acid releasing agent fully mixes containing JC virus liquid 100 μ L, more centrifugal 5min, 12000rpm, get supernatant liquor spectrophotometric measurement A260 quantitative, be then diluted to 1.0 × 10
6copy/mL, namely can be used as positive quality control product template.
Particularly, critical positive quality control product is 1.0 × 10
4the DNA fragmentation solution of the JC virogene of copy/mL.Get containing JC virus liquid, strain is provided by China typical culture collection center (CCTCC), after cultivating, get and add isopyknic nucleic acid releasing agent containing JC virus liquid 100 μ L, after abundant mixing, more centrifugal 5min, 12000rpm, get supernatant liquor spectrophotometric measurement A260 quantitative, be then diluted to 1.0 × 10
4copy/mL, namely can be used as critical positive quality control product.
Particularly, working standard comprises: working standard 1, working standard 2, working standard 3 and working standard 4, wherein,
Working standard 1 is for containing 1.0 × 10
7the non-infectious DNA fragmentation of the JC virogene of copy/mL;
Working standard 2 is for containing 1.0 × 10
6the non-infectious DNA fragmentation of the JC virogene of copy/mL;
Working standard 3 is for containing 1.0 × 10
5the non-infectious DNA fragmentation of the JC virogene of copy/mL;
Working standard 4 is for containing 1.0 × 10
4the non-infectious DNA fragmentation of the JC virogene of copy/mL.
Working standard, for the pUC57-T recombinant plasmid of the nucleotide fragments of the high conservative gene containing JC virus, alkaline lysis method of extracting DNA is used after this recombinant plasmid transformed bacillus coli DH 5 alpha propagation, through DNA Purification Kit, with spectrophotometric measurement A260 quantitative, be then diluted to 1.0 × 10 according to formula scales
8copy/mL, in-20 DEG C of preservations.Stock concentration is 1.0 × 10
8copy/mL, uses front stroke-physiological saline solution or 0.1mol/L PBS damping fluid (pH value is 7.6) to carry out the serial dilution of 10 times of gradients.Working concentration is followed successively by 1.0 × 10
7copy/mL, 1.0 × 106 copies/mL, 1.0 × 10
5copy/mL and 1.0 × 10
4copy/mL, before using, through the centrifugal 30s of 10,000rmp, gets supernatant liquor as working standard.The Kit components that the present embodiment provides is as follows:
Table 2. test kit configuration (24 person-portions/box):
This test kit can store-10 DEG C ± 5 DEG C lucifuges, avoid multigelation, the quantitative real time PCR Instrument that this test kit is suitable for comprises: Roche LightCycler480, ABI7500, ABI7300, Bio-Rad iQ5TM, Stratagene Mx3000P, Stratagene Mx3005P and Da An 7000 etc.
The test kit provided by the embodiment of the present invention 2 detects JC virus on Bio-Rad iQ5TM quantitative real time PCR Instrument, and concrete grammar is as follows:
(1) collected specimens: sample all derives from Wuhan Infectious Diseases Hospital, wherein 5 is positive known after testing, 3 is negative sample known after testing, sample can be blood plasma, serum or urine, and in 5 known after testing positive, have 2 samples to be serum, 1 is blood plasma, all the other 2 is urine, in 3 known after testing negative samples, have 1 sample to be serum, 1 is blood plasma, and all the other 1 is urine.
1. the pre-treatment of serum: get 0.5ml blood product and be placed in 1.5ml centrifuge tube, the centrifugal 5min of 2000r/min, isolates serum.Centrifuge tube 4 DEG C saves backup.
2. the pre-treatment of blood plasma: get 0.5ml blood product and be placed in 1.5ml centrifuge tube, add antithrombotics, put upside down mixing gently, the centrifugal 5min of 2000r/min, isolates blood plasma.Centrifuge tube 4 DEG C saves backup.
3. urine mark product process: by the urine sample vibration mixing gathered, 1ml is transferred in centrifuge tube, and the centrifugal 10min of 13000rpm, abandons supernatant, and centrifuge tube 4 DEG C saves backup.
Sample retention and transport: if sample is not tested immediately, should be stored in-20 DEG C, avoid multigelation.The long-distance transport of sample should adopt 0 DEG C of curling stone.
(2) sample preparation: get after testing sample and equivalent volumes DNA extraction liquid fully mixes, be the sample after process.
(3) application of sample: add each 0.5 μ L of the sample after process, negative quality control product, positive quality control product, critical positive quality control product and working standard respectively in the PCR reaction tubes that PCR reaction solution, primer and probe be housed, the volume ratio of PCR reaction solution and sample, negative quality control product, positive quality control product, critical positive quality control product or working standard after processing is 99:1, through the centrifugal 10s of 5,000rpm.
(4) pcr amplification: the reactive tank each reaction tubes being put into quantitative fluorescent PCR instrument, arranges mark fluorescent radical species, sample ID and type, definition sample well: negative quality control product selects NTC; The choosing of measuring samples, positive quality control product and critical positive quality control product select Unknown.
Pcr amplification is carried out by program below:
95 DEG C 3 minutes;
95 DEG C 10 seconds, 60 DEG C 1 minute (collection fluorescent signal), 40 circulations.
(5) judgement is analyzed:
Ct value be less than 28 for positive; Ct value be greater than 32 for negative; Ct value be more than or equal to 28 and be less than or equal to 32 for the critical positive.The amplification that the embodiment of the present invention provides is (Fig. 1 can judge sample result when Ct value is 32) as shown in Figure 1, and concrete detected result sees the following form 3.
The Ct value that the sample that table 3 provides for embodiment 2 is corresponding
| Sequence number | Ct value |
| 1 | 26.79 |
| 2 | N/A |
| 3 | N/A |
| 4 | 21.38 |
| 5 | 24.32 |
| 6 | 20.78 |
| 7 | 20.08 |
| 8 | N/A |
Wherein, sample Isosorbide-5-Nitrae, 5,6,7 in positive scope, is positive; N/A represents negative, sample 2,3,8 in negative range, is negative sample, result is consistent with known, the test kit specificity that the visible embodiment of the present invention 2 provides is 100%, and positive rate is 100%, for Fig. 1, marked by positive amplification in FIG, the arrangement rule in Fig. 2-Fig. 5 is see Fig. 1.
Embodiment 3. 1 kinds is for detecting the test kit of JC virus
Embodiments provide a kind of test kit detecting JC viral nucleic acid in sample, the composition in the test kit provided in the composition of this test kit and embodiment 2 is distinguished and is:
The proportioning of each component concentration of PCR reaction solution is: concentration is the Taq enzyme 0.1 μ L of 5U/ μ L, concentration is 10mmol/L dNTPs 1 μ L, 10 × PCR Buffer 5 μ L and concentration are the MgCl of 25mmol/L
2solution 3 μ L.
Concentration is that the forward primer of 10 μm of ol/L and reverse primer respectively add 0.25 μ L, and concentration is that 10 μm of ol/L probes add 0.25 μ L.
In actual application, primer and probe together can be added in PCR reaction solution, then to add sterilized water to volume be 10 μ L.
DNA extraction liquid comprises that Triton-X100 final concentration is 0.1%, NP-40 final concentration be 0.2% and Tris-HCL (pH value is 8.3) final concentration be 0.05M/L.
Test kit configuration (24 person-portions/box) that table 4. embodiment 3 provides:
Collected specimens: sample all derives from Wuhan Infectious Diseases Hospital, wherein 8 is positive known after testing, and 2 is negative sample known after testing.Other steps are see embodiment 2, and difference is:
Application of sample: add the sample after process, negative quality control product, positive quality control product and critical positive quality control product 40 μ L respectively in the PCR reaction tubes that 10 μ L PCR reaction solutions are housed, build pipe lid, the centrifugal 10s of 5,000rpm.
Operated according to the step in embodiment 2 by above-mentioned 10 samples, the amplification that the embodiment of the present invention provides as shown in Figure 2, obtains concrete detected result in table 5.
The Ct value that the sample that table 5 provides for embodiment 3 is corresponding
| Sequence number | Ct value |
| 1 | 29.55 |
| 2 | 23.79 |
| 3 | 18.56 |
| 4 | N/A |
| 5 | 22.24 |
| 6 | 24.66 |
| 7 | N/A |
| 8 | 22.46 |
| 9 | 21.27 |
| 10 | 30.69 |
In table 5, sample 2,3,5,6,8 and 9, in positive scope, is positive; Sample 1 and 10, in critical positive scope, is critical positive, positive totally 8 and conform to known detected result; Sample 4 and 7, in negative range, is negative sample, conforms to known detected result, and the test kit specificity that the visible embodiment of the present invention 3 provides is 100%, and positive rate is 100%.
Embodiment 4. 1 kinds is for detecting the test kit of JC virus
Embodiments provide a kind of test kit detecting JC viral nucleic acid in sample, the composition in the test kit provided in the composition of this test kit and embodiment 2 is distinguished and is:
The proportioning of each component concentration of PCR reaction solution is: concentration is the Taq enzyme 0.5 μ L of 5U/ μ L, concentration is 10mmol/L dNTPs 3 μ L, 10 × PCR Buffer 5 μ L and concentration are the MgCl of 25mmol/L
2solution 10 μ L.
Concentration is that the forward primer of 10 μm of ol/L and reverse primer respectively add 4.5 μ L, and concentration is that 10 μm of ol/L probes add 4.5 μ L.
In actual application, primer and probe together can be added in PCR reaction solution, then to add sterilized water to volume be 45 μ L.
DNA extraction liquid comprises that Triton-X100 final concentration is 0.1%, NP-40 final concentration be 0.2% and Tris-HCL (pH value is 8.3) final concentration be 0.05M/L.
The Reagent evaluation provided in all the other reagent and embodiment 2 with.
Collected specimens: sample all derives from Wuhan Infectious Diseases Hospital, wherein 6 is positive known after testing, and 2 is negative sample known after testing.Other steps are see embodiment 2, and difference is:
Application of sample: add each 5 μ L of the sample after process, negative quality control product, positive quality control product and critical positive quality control product respectively in the PCR reaction tubes that 10 μ L PCR reaction solutions are housed, the volume ratio of PCR reaction solution and sample, negative quality control product, positive quality control product or critical positive quality control product after processing is 9:1, build pipe lid, the centrifugal 10s of 5,000rpm.
Operated according to the step in embodiment 2 by above-mentioned 10 samples, the amplification that the embodiment of the present invention provides as shown in Figure 3, obtains concrete detected result in table 6.
The Ct value that the sample that table 6 provides for embodiment 4 is corresponding
| Sequence number | Ct value |
| 1 | 24.11 |
| 2 | 30.17 |
| 3 | 22.53 |
| 4 | 31.59 |
| 5 | N/A |
| 6 | N/A |
| 7 | 26.01 |
| 8 | 31.55 |
In table 6, sample 1,3 and 7, in positive scope, is positive; Sample 2,4 and 8, in critical positive scope, is critical positive, positive totally 6 and conform to known detected result; Sample 5 and 6, in negative range, is negative sample, conforms to known detected result, and the test kit specificity that the visible embodiment of the present invention 4 provides is 100%, and positive rate is 100%.
Embodiment 5. 1 kinds is for detecting the test kit of JC virus
When the test kit utilizing the embodiment of the present invention 4 to provide carries out detection by quantitative, need drawing standard curve, except 8 example reaction pipes, separately get 3 reaction tubess and be respectively negative quality control product, positive quality control product and critical positive quality control product, also have 4 reaction tubess, correspondence adds the working standard 5 μ L of different concns gradient in test kit, according to the method preparation PCR reaction system of embodiment 4, the centrifugal 10s of 5000rpm, then puts into instrument sample groove and carries out pcr amplification.Working standard selects Standard.For Standard, need to input 1.0 × 10 respectively in Quantity hurdle
7copy/ml, 1.0 × 10
6copy/ml, 1.0 × 10
5copy/ml, 1.0 × 10
4copy/ml and 1.0 × 10
3copy/ml.
Adopt and use instrument Bio-Rad iQ5TM to detect.
Reference results:
If a. not S-type or Ct value > 32 of amplification curve, judge that sample JC viral DNA content is less than Monitoring lower-cut;
If b. not obvious or 28≤Ct value≤32 of amplification curve S type, then sample JC viral DNA content is in critical positive scope;
If the c. S-type and Ct value ﹤ 28 of amplification curve, then carry out quantitatively by the following method: if the C of sample (" C " represents sample concentration or content) < 5.0000E+01, then JC viral DNA total content < 50 gene copy of this sample; If the 5.0000E+01≤C of sample≤5.0000E+07, then the JC viral DNA total content of this sample equals C gene copy; If the C > 5.0000E+07 of sample, then the JC viral DNA total content > 5.0000E+07 gene copy of this sample, detect in diluted sample to linearity range, concrete detected result is in table 7 again;
The initial concentration that table 7 is Ct value and correspondence thereof
| Working standard | Ct value | C (initial concentration) |
| 1.0e+007 | 18.30 | 1.0e+007 |
| 1.0e+006 | 21.62 | 1.0e+006 |
| 1.0e+005 | 24.94 | 1.0e+005 |
| 1.0e+004 | 28.26 | 1.0e+004 |
| Slope | -3.32 | - |
| Intercept | 41.54 | - |
| R 2 | 1 | - |
| Standard equation | y＝-3.32x+41.54 | - |
The amplification curve of the sample that the embodiment of the present invention 5 provides as shown in Figure 5, the working standard that the embodiment of the present invention 5 provides detects together with 8 samples, according to the Ct value obtained after amplification, looks into the typical curve of Fig. 6, again through converting, finally obtain the starting point concentration of 8 samples as following table.
| Sequence number | Ct value | C (initial concentration) |
| Positive quality control product | 19.68 | 3.840e+006 |
| Critical positive quality control product | 29.27 | 4.960e+003 |
| Negative quality control product | N/A | - |
| 1 | 26.01 | 4.761e+004 |
| 2 | 25.43 | 7.118e+004 |
| 3 | 34.24 | 1.580e+002 |
| 4 | 22.42 | 5.742e+005 |
| 5 | N/A | - |
| 6 | N/A | - |
| 7 | N/A | - |
| 8 | 26.03 | 4.696e+004 |
Amplification curve is all in smooth " S " type, and typical curve is straight line, and Ct value is between 14-28, and the Ct value difference of each concentration gradient is about 3.32.Monitoring lower-cut can be accurate to 5.000e+002, and as can be seen here, this test kit has highly sensitive.
The present invention relates to a kind of pathogen gene detection technique causing the diseases such as mankind's progressive multifocal leukoencephalopathy, be applicable to JC virus qualitative and quantitative detection.The real-time TaqMan quantitative fluorescent PCR that the embodiment of the present invention provides, its primer and fluorescent probe have high specific and highly sensitive, accurately can detect sample and whether infect JC virus, and its test kit can accurate quantification, and can detect JC virus fast and accurately.Real-time TaqMan quantitative fluorescent PCR detects target gene with stopped pipe pattern by sequence-specific TaqMan fluorescent probe while amplification, thus can increase the possibility of specificity and reduction crossed contamination.In addition, the embodiment of the present invention does not need further downstream analysis, has saved the time of gel electrophoresis observations, and whole testing process only needs 2 ~ 3 hours altogether.In real-time TaqMan quantitative fluorescent PCR, each circulation of PCR primer accumulation is monitored in real time and is analyzed, and analyzes the cycle number (Ct value) reaching fluorescence threshold and just can directly report out DNA starting copy number.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. for detecting primer and the probe of JC virus, it is characterized in that, described primer and probe comprise: for detect JC virus forward primer, for detecting the reverse primer of JC virus and the probe for detecting JC virus, wherein,
The described forward primer for detecting JC virus is as shown in SEQ ID NO.1 in sequence table;
The described reverse primer for detecting JC virus is as shown in SEQ ID NO.2 in sequence table;
The described probe for detecting JC virus is as shown in SEQ ID NO.3 in sequence table;
5 ' end of described probe is connected with fluorophor FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy5, and 3 ' end of described probe is connected with quenching group TAMRA, Eclipse, BHQ1, BHQ2, BHQ3 or DABCYL.
2. for detecting a test kit for JC virus, it is characterized in that, described test kit comprises primer as claimed in claim 1 and probe.
3. test kit according to claim 2, is characterized in that, described test kit also comprises: nucleic acid releasing agent, PCR reaction solution, critical positive quality control product, positive quality control product, negative quality control product and working standard.
4. test kit according to claim 3, is characterized in that, described working standard comprises: working standard 1, working standard 2, working standard 3 and working standard 4, wherein,
Described working standard 1 is 1 × 10
7the gene fragment of the described JC virus of copy/mL;
Described working standard 2 is 1 × 10
6the gene fragment of the described JC virus of copy/mL;
Described working standard 3 is 1 × 10
5the gene fragment of the described JC virus of copy/mL;
Described working standard 4 is 1 × 10
4the gene fragment of the described JC virus of copy/mL.
5. test kit according to claim 3, is characterized in that, described positive quality control product is 1.0 × 10
6the T antigen gene fragment of the described JC virus of copy/mL.
6. test kit according to claim 3, is characterized in that, the final concentration of described forward primer and described reverse primer is 0.05-0.9 μM, and the final concentration of described probe is 0.05-0.9 μM.
7. test kit according to claim 3, it is characterized in that, each component of described PCR reaction solution comprises in pcr amplification reaction system: dNTPs, 10 × PCR damping fluid and final concentration that the Taq enzyme that final concentration is 0.01U/ μ L ~ 0.05U/ μ L, final concentration are 0.2 ~ 0.6mM are the solution containing Mg ion of 1.5 ~ 5.0mM.
8. test kit according to claim 3, it is characterized in that, described nucleic acid releasing agent comprises sterilized water, Nonyl pheno (40) ether that Triton X-100 that final concentration is 0.03-0.3%, final concentration are 0.04-0.4% and pH value be 8.3 final concentration be three (methylol) aminomethane of 0.01-0.1M.
9. test kit according to claim 3, is characterized in that, described critical positive quality control product is 1.0 × 10
4the gene fragment of the described JC virus of copy/mL.
10. test kit according to claim 3, is characterized in that, described negative quality control product to be concentration be 0.8% physiological saline.
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| CN201510052911.3A CN104745724A (en) | 2015-01-30 | 2015-01-30 | Primers, probe and kit used for detecting JC viruses (JCVs) |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106048093A (en) * | 2016-08-02 | 2016-10-26 | 北京思尔成生物技术有限公司 | JC virus detection method, kit and application of kit |
| CN107586883A (en) * | 2017-09-27 | 2018-01-16 | 湖北朗德医疗科技有限公司 | A kind of primer, probe and kit for being used to detect JC viruses |
| CN108004348A (en) * | 2017-09-27 | 2018-05-08 | 湖北朗德医疗科技有限公司 | A kind of primer, probe and kit for being used to detect enteric adenovirus |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102690895A (en) * | 2011-07-27 | 2012-09-26 | 中国人民解放军第三〇九医院 | Detection method of JC virus as well as kit and application thereof |
| CN103827319A (en) * | 2011-07-29 | 2014-05-28 | 生物基因Idecma公司 | Assay for detection of jc virus dna |
-
2015
- 2015-01-30 CN CN201510052911.3A patent/CN104745724A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102690895A (en) * | 2011-07-27 | 2012-09-26 | 中国人民解放军第三〇九医院 | Detection method of JC virus as well as kit and application thereof |
| CN103827319A (en) * | 2011-07-29 | 2014-05-28 | 生物基因Idecma公司 | Assay for detection of jc virus dna |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106048093A (en) * | 2016-08-02 | 2016-10-26 | 北京思尔成生物技术有限公司 | JC virus detection method, kit and application of kit |
| CN107586883A (en) * | 2017-09-27 | 2018-01-16 | 湖北朗德医疗科技有限公司 | A kind of primer, probe and kit for being used to detect JC viruses |
| CN108004348A (en) * | 2017-09-27 | 2018-05-08 | 湖北朗德医疗科技有限公司 | A kind of primer, probe and kit for being used to detect enteric adenovirus |
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