CN104745722A - Primers, probe and kit used for detecting varicella zoster viruses (VZVs) - Google Patents
Primers, probe and kit used for detecting varicella zoster viruses (VZVs) Download PDFInfo
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- CN104745722A CN104745722A CN201510050800.9A CN201510050800A CN104745722A CN 104745722 A CN104745722 A CN 104745722A CN 201510050800 A CN201510050800 A CN 201510050800A CN 104745722 A CN104745722 A CN 104745722A
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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
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
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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
- C12Q1/6851—Quantitative amplification
Abstract
The invention discloses primers, probe and kit used for detecting varicella zoster viruses (VZVs), 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 VZVs. The kit comprises the primers and the probe. The primers, the probe and the kit have the characteristic of high sensitivity, and the detection speed is high and the whole detection process only takes 2-3 hours.
Description
Technical field
The present invention relates to biological technical field, particularly a kind of primer, probe and test kit for detecting varicella zoster virus.
Background technology
Varicella zoster virus (Varicella zoster virus, VZV) herpetoviridae α subfamily is belonged to, it is DNA virus, be human herpesvirus 3 type (Human Herpesvirus3 by definite designation at present, HHV-3), its unique host is the mankind, thinks that this virus only has a serotype at present.This virus is the pathogenic agent of varicella and zoster, and not acidproof, thermo-labile, resistibility is more weak to external world, can by ether deactivation, but infectivity is extremely strong, and the people of 90% had just infected VZV before growing up.VZV enters human body through respiratory mucosa and forms primary infection, and clinical manifestation is varicella.One of disease common Childhood that varicella being, infectivity is strong, and in Rash diease, the infectivity of varicella is only second to measles, is more common in less than 10 years old children.
Laboratory diagnostic method is membrane antigen Immunofluorescent Antibody detection experiment (FAMA), FAMA method need gather acute phase and decubation paired sera, the extraction time of sample is longer, makes this detection not reach the object of quick diagnosis.
Summary of the invention
The problem of quick diagnosis can not being realized to solve prior art, embodiments providing a kind of primer, probe and test kit for detecting varicella zoster virus.Described technical scheme is as follows:
On the one hand, embodiments provide a kind of primer for detecting varicella zoster virus and probe, described primer and probe comprise: for detect varicella zoster virus forward primer, for detecting the reverse primer of varicella zoster virus and the probe for detecting varicella zoster virus, wherein
For detecting the forward primer of varicella zoster virus as shown in SEQ ID NO.1 in sequence table;
For detecting the reverse primer of varicella zoster virus as shown in SEQ ID NO.2 in sequence table;
For detecting the probe of varicella zoster virus 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 varicella zoster 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 varicella zoster virus of copy/mL;
Described working standard 2 is 1 × 10
6the gene fragment of the described varicella zoster virus of copy/mL;
Described working standard 3 is 1 × 10
5the gene fragment of the described varicella zoster virus of copy/mL;
Described working standard 4 is 1 × 10
4the gene fragment of the described varicella zoster virus of copy/mL.
Particularly, described positive quality control product is 1.0 × 10
6the gene fragment of the described varicella zoster 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, described nucleic acid releasing agent comprises: Nonyl pheno (40) ether that sterilized water, final concentration are the Triton X-100 of 0.03-0.3%, final concentration is 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 varicella zoster 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 varicella zoster virus that the embodiment of the present invention provides, there is highly sensitive characteristic, the test kit for detecting varicella zoster virus that the embodiment of the present invention is provided, there is highly sensitive characteristic, simultaneously, detection speed is fast, whole testing process only needs 2 ~ 3 hours altogether, 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.
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.
The primer that embodiment 1. 1 kinds detects for varicella zoster virus nucleic acid quantification and probe
Embodiments provide a kind of primer for detecting varicella zoster virus and probe, primer and probe comprise: for detect varicella zoster virus forward primer, for detecting the reverse primer of varicella zoster virus and the probe for detecting varicella zoster virus, wherein
For detecting the forward primer of varicella zoster virus as shown in SEQ ID NO.1 in sequence table;
For detecting the reverse primer of varicella zoster virus as shown in SEQ ID NO.2 in sequence table;
For detecting the probe of varicella zoster 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. varicella zoster virus primer and probe
In table 1: F:forward, forward; Varicella zoster virus-F represents the forward primer of varicella zoster virus.R:reverse, oppositely; Varicella zoster virus-R represents the reverse primer of varicella zoster virus.P:probe, fluorescent probe (probe); Varicella zoster virus-P represents varicella zoster virus fluorescent probe, and fluorescent probe can be TaqMan-MGB fluorescent probe, LNA fluorescent probe or MGB fluorescent probe.Primer in 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 varicella zoster virus in testing sample.
Embodiment 2. 1 kinds is for detecting the test kit of varicella zoster virus
Embodiments provide a kind of test kit for detecting varicella zoster virus nucleic acid quantification, 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 is the physiological saline of concentration 0.8%.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 varicella zoster virus gene fragment of copy/mL.Get containing varicella zoster virus liquid, strain is by China typical culture collection center (China Center for Type CultureCollection, CCTCC) provide, after cultivating, get and add after isopyknic nucleic acid releasing agent fully mixes containing varicella zoster 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 comprises containing concentration is 1.0 × 10
4the DNA fragmentation solution of the varicella zoster virus gene of copy/mL.Get containing varicella zoster virus liquid, strain is provided by China typical culture collection center (CCTCC), after cultivating, get and add isopyknic nucleic acid releasing agent containing varicella zoster 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, and wherein, working standard 1 is 1.0 × 10
7the non-infectious DNA fragmentation of the varicella zoster virus gene of copy/mL;
Working standard 2 is 1.0 × 10
6the non-infectious DNA fragmentation of the varicella zoster virus gene of copy/mL;
Working standard 3 is 1.0 × 10
5the non-infectious DNA fragmentation of the varicella zoster virus gene of copy/mL;
Working standard 4 is 1.0 × 10
4the non-infectious DNA fragmentation of the varicella zoster virus gene of copy/mL.
Working standard, for the pUC57-T recombinant plasmid of the nucleotide fragments of the high conservative gene containing varicella zoster 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 × 10
6copy/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 varicella zoster 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 serum or bleb liquid, in 5 known after testing positive, there are 3 samples to be serum, all the other 2 is bleb liquid, and in 3 known after testing negative samples, have 1 sample to be serum, all the other 2 is bleb liquid.
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, gets supernatant.Sample saves backup in 4 DEG C.
2. bleb liquid sample preparation: take out the sample gathered, add the mixing of 1ml sterilized water, get 0.5ml and be transferred in centrifuge tube, the centrifugal 10min of 13,000rpm, abandons supernatant, and sample saves backup in 4 DEG C.
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 | 24.18 |
| 2 | N/A |
| 3 | N/A |
| 4 | 18.18 |
| 5 | 27.32 |
| 6 | 25.63 |
| 7 | 23.08 |
| 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 varicella zoster virus
Embodiments provide a kind of test kit detecting varicella zoster virus 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 comprise final concentration be 0.15% chloroform, final concentration be 0.25% Virahol and final concentration be the ethanol of 0.36%.
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 LPCR 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 | 23.46 |
| 2 | 18.14 |
| 3 | 24.38 |
| 4 | N/A |
| 5 | 21.38 |
| 6 | 28.55 |
| 7 | N/A |
| 8 | 22.45 |
| 9 | 23.09 |
| 10 | 31.26 |
In table 5, sample 1,2,3,5,8 and 9, in positive scope, is positive; Sample 6 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 varicella zoster virus
Embodiments provide a kind of test kit detecting varicella zoster virus 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 comprise final concentration be 0.3% chloroform, final concentration be 0.4% Virahol and final concentration be the ethanol of 0.6%.
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 LPCR 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 | 27.01 |
| 2 | 31.45 |
| 3 | 27.14 |
| 4 | 30.47 |
| 5 | N/A |
| 6 | N/A |
| 7 | 23.32 |
| 8 | 31.11 |
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 varicella zoster 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 varicella zoster virus DNA content is less than Monitoring lower-cut;
If b. not obvious or 28≤Ct value≤32 of amplification curve S type, then sample varicella zoster virus 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 varicella zoster virus DNA total content < 50 gene copy of this sample; If the 5.0000E+01≤C of sample≤5.0000E+07, then the varicella zoster virus DNA total content of this sample equals C gene copy; If the C > 5.0000E+07 of sample, then the varicella zoster virus 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 | 22.12 | 1.0e+007 |
| 1.0e+006 | 25.51 | 1.0e+006 |
| 1.0e+005 | 28.78 | 1.0e+005 |
| 1.0e+004 | 32.10 | 1.0e+004 |
| Slope | -3.322 | - |
| Intercept | 45.395 | - |
| R 2 | 1.000 | - |
| Standard equation | y＝-3.322x+45.395 | - |
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 | 23.61 | 3.612e+006 |
| Critical positive quality control product | 29.98 | 4.368e+004 |
| Negative quality control product | N/A | — |
| 1 | 22.72 | 6.694e+006 |
| 2 | 26.18 | 6.084e+005 |
| 3 | 29.57 | 5.804e+004 |
| 4 | 24.14 | 2.502e+006 |
| 5 | 36.97 | 3.437e+002 |
| 6 | N/A | — |
| 7 | 31.77 | 1.263e+004 |
| 8 | N/A | — |
Amplification curve is all in smooth " S " type, and typical curve is straight line.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 Human Varicella, be applicable to varicella zoster 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, and detection speed is fast, its test kit can accurate quantification, this test kit can detect varicella zoster virus fast and accurately, and whole testing process only needs 2 ~ 3 hours altogether.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.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. one kind for detecting primer and the probe of varicella zoster virus, it is characterized in that, described primer and probe comprise: for detect varicella zoster virus forward primer, for detecting the reverse primer of varicella zoster virus and the probe for detecting varicella zoster virus, wherein
For detecting the forward primer of varicella zoster virus as shown in SEQ ID NO.1 in sequence table;
For detecting the reverse primer of varicella zoster virus as shown in SEQ ID NO.2 in sequence table;
For detecting the probe of varicella zoster virus 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 varicella zoster 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 varicella zoster virus of copy/mL;
Described working standard 2 is 1 × 10
6the gene fragment of the described varicella zoster virus of copy/mL;
Described working standard 3 is 1 × 10
5the gene fragment of the described varicella zoster virus of copy/mL;
Described working standard 4 is 1 × 10
4the gene fragment of the described varicella zoster 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 gene fragment of the described varicella zoster 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: Nonyl pheno (40) ether that sterilized water, final concentration are the Triton X-100 of 0.03-0.3%, final concentration is 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 varicella zoster virus of copy/mL.
10. test kit according to claim 3, is characterized in that, described negative quality control product is the physiological saline of concentration 0.8%.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510050800.9A CN104745722A (en) | 2015-01-30 | 2015-01-30 | Primers, probe and kit used for detecting varicella zoster viruses (VZVs) |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510050800.9A CN104745722A (en) | 2015-01-30 | 2015-01-30 | Primers, probe and kit used for detecting varicella zoster viruses (VZVs) |
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| CN104745722A true CN104745722A (en) | 2015-07-01 |
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| CN201510050800.9A Pending CN104745722A (en) | 2015-01-30 | 2015-01-30 | Primers, probe and kit used for detecting varicella zoster viruses (VZVs) |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109913589A (en) * | 2019-04-09 | 2019-06-21 | 深圳市儿童医院 | It is a kind of for detecting primer combination of probe object, kit and the method for herpes zoster virus |
| CN111074003A (en) * | 2020-01-10 | 2020-04-28 | 上海润达榕嘉生物科技有限公司 | VZ virus detection primer and kit thereof |
| CN111088401A (en) * | 2020-01-10 | 2020-05-01 | 上海润达榕嘉生物科技有限公司 | Multi-virus detection primer and kit thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101871013A (en) * | 2009-04-22 | 2010-10-27 | 中山大学达安基因股份有限公司 | Kit for detecting varicella-herpes zoster virus |
| CN103866046A (en) * | 2014-01-21 | 2014-06-18 | 浙江大学 | Detection kit for human herpes viruses EBV and VZV |
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| CN101871013A (en) * | 2009-04-22 | 2010-10-27 | 中山大学达安基因股份有限公司 | Kit for detecting varicella-herpes zoster virus |
| CN103866046A (en) * | 2014-01-21 | 2014-06-18 | 浙江大学 | Detection kit for human herpes viruses EBV and VZV |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109913589A (en) * | 2019-04-09 | 2019-06-21 | 深圳市儿童医院 | It is a kind of for detecting primer combination of probe object, kit and the method for herpes zoster virus |
| CN111074003A (en) * | 2020-01-10 | 2020-04-28 | 上海润达榕嘉生物科技有限公司 | VZ virus detection primer and kit thereof |
| CN111088401A (en) * | 2020-01-10 | 2020-05-01 | 上海润达榕嘉生物科技有限公司 | Multi-virus detection primer and kit thereof |
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