CN106148564A - A kind of method utilizing digital pcr detection circovurus type 2 viral level - Google Patents
A kind of method utilizing digital pcr detection circovurus type 2 viral level Download PDFInfo
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- CN106148564A CN106148564A CN201510107633.7A CN201510107633A CN106148564A CN 106148564 A CN106148564 A CN 106148564A CN 201510107633 A CN201510107633 A CN 201510107633A CN 106148564 A CN106148564 A CN 106148564A
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- digital pcr
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Abstract
The present invention relates to a kind of method utilizing circovurus type 2 viral level in drop formula digital pcr detection sample and test kit, belong to virus detection techniques field.The method includes 1) extraction of porcine circovirus genomic DNA；2) configuration of ddPCR reaction system, carries out digital pcr reaction；3) according to the signal of reaction, testing result is carried out interpretation.This method, without arranging standard curve, can immediately arrive at circovurus type 2 genomic DNA copy number in sample according to testing result, it is achieved that the absolute quantitation of nucleic acid copies.
Description
Technical field
The present invention relates to field of biological genes, relate to a kind of method that viral nucleic acid is quantitative, utilize digital pcr to detect porcine circovirus particularly to one
The method of 2 type nucleic acid contents.
Background technology
Porcine circovirus desease is a kind of multisystem functional disorder disease of the pig caused by pig circular ring virus (PCV), congenital with newborn piglet clinically
Tremble and postweaning multisystemic asthenia syndrome is cardinal symptom.Pig circular ring virus (porcine circovirus, PCV) belongs on taxonomy
Porcine circovirus section Circovirus, is one of minimum animal virus.Know that PCV has two serotypes, i.e. PCV1 and PCV2.PCV1 is non-
Pathogenic virus, PCV2 is pathogenic virus.For PCV2 qualification and detection had many methods, as electron microscopic observation, ELISA, IFA,
PCR, quantitative fluorescent PCR (qPCR) etc., qPCR technology has the features such as quick, sensitive, high specific in the above-mentioned methods, is therefore more come
The most detections being applied to various virus.But, qPCR's is quantitatively " relative quantification ", and its accuracy and repeatability still can not meet
The requirement of molecular biology quantitative analysis.
(PCR (digitalpolymerase chain reaction, dPCR) is one and detects and the new technique of quantitative nucleic acid digital pcr.20 generation
Ji Mo, Vogelstein etc. propose the concept of dPCR, by a sample is divided into tens to several ten thousand parts, are assigned to different reaction members, often
Individual unit comprises the target molecule (DNA profiling) of one or more copy, respectively target molecule is carried out PCR amplification in each reaction member,
Amplification carries out statistical analysis to the fluorescence signal of each reaction member after terminating.Unlike qPCR, dPCR does not relies on CT value, therefore
Do not affected by amplification efficiency, expanded mean concentration (content) energy being calculated each reaction member after terminating by directly counting or Poisson distribution formula
Enough by error control within 5%, dPCR can need not reference standard sample and standard curve to realize absolute quantification analysis.Owing to dPCR is
One excellent technique of quantitative minim DNA molecule with repeatability. it is easy to operate, detection flux height, high specificity, highly sensitive, fixed
The amount advantage such as accurately has become the important tool in molecular biology research, and has been applied to single cell analysis, early diagnosis of cancer and antenatal
The research fields such as diagnosis, epidemic disease detection.DPCR technology is used for the detection of PCV2, and it is the most fixed to carry out PCV2 nucleic acid fast and accurately
Amount.
Summary of the invention:
The mesh of the present invention is to provide a kind of test kit utilizing PCV2 nucleic acid concentration in digital pcr detection sample and method.
The application in animal epidemic detection, production of vaccine quality control of the digital pcr method falls within protection scope of the present invention.
The present invention is a kind of method of highly sensitive detection circovurus type 2, and step includes:
(1) extraction of viral DNA in sample, it is thus achieved that PCR expands template；
(2) configuration of digital pcr reaction system, carries out dPCR reaction；Described PCR reaction system expands mould containing the PCR that (1) obtains in steps
Plate, PCV2 special primer to, probe, dNTP and archaeal dna polymerase；
(3) fluorescence signal produced according to step (2) dPCR reaction, the PCV2 copy number being calculated in described testing sample；
In the above-mentioned methods, described testing sample can be tissue pathological material of disease, can also be virocyte culture.In an embodiment of the present invention, institute
State testing sample for tissue pathological material of disease, specially pig lymph node, spleen and kidney；
(4) when described measuring samples is for tissue pathological material of disease, in the step (1) of said method, described " extraction of viral DNA " can be by described to be measured
Sample (tissue pathological material of disease) is ground homogenate, freeze thawing 3 times.Described testing sample (tissue pathological material of disease) 8000～14000g is centrifuged 1～5min,
Temperature can be 4 DEG C.Take supernatant, use commercialization viral DNA to extract test kit and carry out nucleic acid extraction.
When above-mentioned sample is the fluid samples such as blood, tissue fluid, urine, in said method step (1), described " extraction of viral DNA "
Can be 8000～14000g to be centrifuged 1～5min, use commercialization viral DNA to extract test kit and carry out nucleic acid extraction.
In the step (2) of above-mentioned detection method, annealing temperature when carrying out the reaction of described digital pcr can be 53 DEG C.Further, in the present invention
In, response procedures when carrying out the reaction of described digital pcr is: 95 DEG C of 10min；94 DEG C of 30s, 53 DEG C of 60s, 40 circulations；98℃10
min.PCR instrument warming and cooling rate is set as≤2.5 DEG C.
In the step (2) of above-mentioned detection method, described optimal reaction system is 2 × ddPCR supermix for probe 10uL, upstream and downstream
The each 100-900nmol/L of primer, probe 100-900nmol/L, template DNA (cDNA) is less than 200ng, benefit aquesterilisa to cumulative volume 20uL；
In the step (2) of above-mentioned detection method, described PCV2 primer is specially by two single stranded DNAs shown in sequence in sequence table 1 and sequence 2
Molecule primers pair, probe is 1 shown in sequence 3 single strand dna in sequence table.
In the above-mentioned methods, the fluorophor of described probe is selected from 6-CF 5(6)-Carboxyfluorescein (FAM), chlordene-6-carboxyl fluorescence number (HEX), Cy5
(U.S. Life Technologies), Cy3 (U.S. Life Technologies), VIC (U.S. Life Technologies), fluorescence is quenched
The group that goes out is selected from 6-carboxyl tetramethylrhodamin (TAMARA), BHQ1 (U.S. Life Technologies), BHQ2 (U.S. Life Technologies)
Or MGB (U.S. Life Technologies).
In the above-mentioned methods, carry out described digital pcr reaction, and calculate PCV2 copy nucleic acid in described testing sample according to the fluorescence signal produced
Number.First, utilize the drop generator of digital pcr system that the reaction system configured carries out separatory process, generate 10000～20000 oil
The reaction drop of Bao Shui, makes the PCR amplification template number of each microdroplet be less than or equal to 1；Secondly, carry out PCR cycle amplification and carry out result
Judge: the product after pcr amplification reaction is carried out signal collection, determines PCV2DNA template according to the amount of droplets having fluorescence signal in drop
Quantity.
Available QuantaSoft (Bio-Rad) software carries out data analysis, calculates the nucleic acid copies of virus in sample.
By said method, the viral nucleic acid copy number of PCV2 can be detected.
The beneficial effects of the present invention is: the present invention uses digital pcr technology for detection PCV2 virus, the sensitivity of its detection is slightly above fluorescent quantitation
Round pcr；And this method need not arrange standard curve, according to testing result can the direct nucleic acid copies of PCV2 in interpretation sample, greatly
Simplify operating procedure.
Accompanying drawing explanation
In order to make the object, technical solutions and advantages of the present invention clearer, below in conjunction with accompanying drawing, the present invention is described in further detail,
Wherein:
Fig. 1 dPCR annealing temperature optimum results (A～H is followed successively by 61 DEG C～51 DEG C)
Fig. 2 dPCR each Concentraton gradient testing result.Standard concentration is 4.85 × 103copy/ul、4.85×102copy/ul、4.85×101copy/ul、4.85×
10-1copy/ul、2.43×10-1copy/ul、1.21×10-1copy/ul、6.06×10-2copy/ul。
Fig. 3 dPCR each Concentraton gradient testing result linear relationship chart.
Detailed description of the invention:
The present invention will be further described with instantiation below in conjunction with the accompanying drawings, but not as a limitation of the invention.
1. main experimental instrument
CFX96 quantitative real time PCR Instrument (Bio-Rad company)；Horizontal strip electrophoresis and Labworks image acquisition and analysis software (Bio-Rad company)；Grads PCR expands
Increase instrument (Bio-Rad company)；Ultramicrospectrophotometer；QX-i00 droplet type digital pcr instrument (Bio-Rad company)；Thermo instrument for extracting nucleic acid；
Incubator；Cryogenic refrigerator；Thermostatical circulating water bath；High speed refrigerated centrifuge；Room temperature centrifuge；
2 test materials and reagent
Viral DNA/RNA paramagnetic particle method extracts test kit (Chengdu Tuo Yang Science and Technology Ltd.)；2×Premix Ex Taq Probe qPCR(TakaRa
Company)；2 × SsoFastTMProbes Supermix (Bio Rad Laboratories)；DNA molecular amount standard, agarose (precious biological engineering (Dalian)
Company limited)；Other reagent are domestic analytical pure.Porcine circovirus vaccine strain is provided by derivatives technology company of Sichuan China.Primer and probe are served
Sea base health bio tech ltd synthesizes, and see table.
| Title | Sequence | Length bp |
| Seq 1 | CGCTGGAGAAGGAAAAATGG | 20 |
| Seq 2 | CTTGACAGTATATCCGAAGGT | 21 |
| Seq 3 | FAM-TTCAACACCCGCCTCTCCCG-BHQ | 20 |
The preparation of 3 viral DNA templates
Viral DNA extracts and uses the viral DNA/RNA nucleic acid extraction kit of Chengdu Tuo Yang Science and Technology Ltd. to operate by operating instruction.Sick
Poison DNA is dissolved in 100 μ l sterile purified waters, uses immediately or-20 DEG C.
The detection program of 4 droplet type digital pcrs
The configuration of 4.1 reactant liquors
Use the 20uL system that 2 × ddPCR supermix for probe (Bio-Rad) reagent is recommended, the template obtained with 2uL step 3,
Add 10uL2 × ddPCR supermix for probe, add each 2uL of upstream and downstream primer (final concentration of 900nmol/L), add probe 2uL
(final concentration of 250nmol/L), unsterilised water to cumulative volume 20uL.
The process of 4.2 reactant liquors
20uL example reaction system is added in 8 holes of the middle row of DG8cartridge, it is to avoid introduce bubble, at DG8cartridge
In lowermost 8 holes, each 70uL microdroplet that adds generates oil (DG oil), covers rubber cushion, above holder puts into microdroplet and generates instrument, generate
Microdroplet.With the volley of rifle fire, the microdroplet of generation slowly proceeded to 96 orifice plate relevant positions, sealer.
4.3 PCR amplifications
96 orifice plates sealing film are proceeded to carry out in grads PCR instrument PCR amplification.Response procedures is 95 DEG C, 10min；94 DEG C, 30s；52 DEG C,
60s, 40 circulations；98 DEG C, 10min).
The reading of 4.4 fluorescence signals
96 orifice plates are put into digital pcr microdroplet fluorescence detector after terminating and are carried out phosphor collection by PCR, send the micro-of fluorescence according in every tube reaction system
Drip quantity, calculate the PCV2 nucleic acid copies in reaction system according to Poisson's law.
Test, simultaneously with real-time fluorescence PCR technology, uses standard curve method, PCV2 is carried out relative quantification.Calculate PCV2 nucleic acid in sample to copy
Shellfish number.
Test in triplicate, results averaged.
Testing result see table:
Claims (6)
1. digital pcr method application in detection by quantitative circovurus type 2 viral level.
2. the method utilizing digital pcr detection circovurus type 2 viral level, including following operating procedure:
(1) porcine circovirus DNA is extracted, it is thus achieved that PCR expands template；
(2) configuration PCR reaction system, carries out digital pcr reaction；Described digital pcr reaction system comprises PCR amplification template that step (1) obtains, porcine circovirus detection primer to, fluorescent probe, dNTP and DNA get together enzyme；
(3) fluorescence signal produced according to the reaction of step (2) digital pcr, the porcine circovirus quantity being calculated in described testing sample.
3. according to the method described in right 2, it is characterised in that: described porcine circovirus primer is to for being made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2 and the fluorescent probe shown in sequence 3.
4. according to the method described in right 2-3, it is characterised in that: described fluorophor is selected from 6-CF 5(6)-Carboxyfluorescein, chlordene-6-carboxyl fluorescence number, Cy5, Cy3 or VIC, and fluorescent quenching group is selected from 6-carboxyl tetramethylrhodamin, BHQ1, BHQ2 or MGB.
5. utilize a test kit for digital pcr detection porcine circovirus content, including following material:
A () is for expanding forward and the Inverse PCR amplification primer of gene to be detected；
(b) probe that conserved sequence is combined on the DNA profiling of testing gene place.
6. require described test kit according to right 5, it is characterised in that: described fluorophor is selected from 6-CF 5(6)-Carboxyfluorescein, chlordene-6-carboxyl fluorescence number, Cy5, Cy3 or VIC, and fluorescent quenching group is selected from 6-carboxyl tetramethylrhodamin, BHQ1, BHQ2 or MGB.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108977583A (en) * | 2018-08-22 | 2018-12-11 | 中国检验检疫科学研究院 | 3 type droplet digital pcr detection primer of pig circular ring virus and probe and its application |
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| CN102382900A (en) * | 2010-08-31 | 2012-03-21 | 上海市农业科学院 | Real-time fluorescence quantitative PCR (polymerase chain reaction) detection method for porcine circovirus type 2 |
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Cited By (1)
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|---|---|---|---|---|
| CN108977583A (en) * | 2018-08-22 | 2018-12-11 | 中国检验检疫科学研究院 | 3 type droplet digital pcr detection primer of pig circular ring virus and probe and its application |
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Application publication date: 20161123 |