CN104450964A - Fluorescent quantitative polymerase chain reaction (PCR) method for detecting mouse poxvirus as well as special primer pair, probe and kit of method - Google Patents
Fluorescent quantitative polymerase chain reaction (PCR) method for detecting mouse poxvirus as well as special primer pair, probe and kit of method Download PDFInfo
- Publication number
- CN104450964A CN104450964A CN201410734124.2A CN201410734124A CN104450964A CN 104450964 A CN104450964 A CN 104450964A CN 201410734124 A CN201410734124 A CN 201410734124A CN 104450964 A CN104450964 A CN 104450964A
- Authority
- CN
- China
- Prior art keywords
- pcr
- probe
- primer
- ect
- dna
- 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.)
- Pending
Links
Classifications
-
- 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
-
- 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
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a fluorescent quantitative polymerase chain reaction (PCR) method for detecting mouse poxvirus as well as special primer pair, a probe and a kit of the method. The base sequence of the primer pair is as follows: an upstream primer: 5'-ACAAGGGGTTGGGTGTAAGA-3' and a downstream primer: 5'-GCGTGCTAGTGGTTGCATTA-3'; the base sequence of the probe is as follows: 5'-R-ACCAAACTGGGTAGACGATGT-Q-3', wherein R represents a fluorescence reporter gene and Q represents a fluorescence quencher gene. The fluorescent quantitative PCR primer pair and the probe disclosed by the invention are quite strong in specificity, and the specific segment of an ERPV-027 gene in the mouse poxvirus can be amplified only; and the detection limit to the mouse poxvirus is 21.7*10<-8>ng/ul, so that detection sensitivity is greatly improved.
Description
Technical field
The invention belongs to animal virus inspection and quarantine technical field, be specifically related to a kind of detect mouse pox virus fluorescence quantifying PCR method and primer special to, probe and test kit.
Background technology
Mousepox (mouse pox) is a kind of deadly infectious disease of the experiment mice caused by mouse pox virus (mouse poxvirus, MPV).This disease is many, and in Outbreak, lethality rate is higher, often causes full group to eliminate, very harmful.Clinical manifestation with four limbs, tail and head swelling, fester, necrosis even toe come off for feature, therefore also known as de-pedopathy (Ectromelia, Ect).
Mouse pox virus belongs to Poxviridae, Chorodopoxvirinae, orthopoxvirus in classification.Nucleic acid based is distrand DNA, and virus particle is oval or brick, diameter 170-250nm.Its genome sequence and variola virus have very large similarity, therefore People's Republic of China's " pharmacopeia " regulation must detect mouse pox virus to mouse biological products, and " laboratory animal microbiology grade and monitoring " national Specification experiment mice must get rid of mouse pox virus.
The infection of mouse pox virus is generally divided into acute infection, chronic infection, latent infection.Wherein, acute infection, chronic infection all more easily find, and the mouse appearance of latent infection mousepox is healthy, without any clinical symptom, virogene is present in certain tissue or cell, do not produce infectious virus, generally not easily detect by current conventional serological antibody detection method, when virus occurs acute attack after being activated under certain conditions.Therefore the mouse group of latent infection is difficult to Timeliness coverage, and hazardness is large.So strengthen inspecting force, improving detection method should cause most attention.
The Serology test of current mouse pox virus is mainly enzyme linked immunosorbent assay, can detect over and infect mousepox or recent infection and the animal survived.But the method there will be false positive to the mice serum that can produce anti-DNA antibody, and can not differentiate that vaccinia virus immune antibody and MPV infect the antibody produced.
Along with the fast development of Protocols in Molecular Biology, polymerase chain reaction (PCR) technology is more and more for laboratory animal disease detection in recent years.Domestic reported first polymerase chain reaction (PCR) technology for detection mouse pox virus in 1998, this detection method is the feature utilizing vaccinia subgroup virus can produce hemagglutinin, according to orthopoxvirus gene conserved regions design pair of primers:
EAC
1：5’-ATG ACA CGA TTG CCA ATAC-3’；
EAC
2：5’-CTA GACTTT GTT TTC TG-3’。
From the serum of mouse pox virus culture and artificial challenge mouse, extract DNA is template, utilizes above-mentioned primer to carry out pcr amplification, carries out electrophoresis detection to amplified production.This method is quick on the draw, and can detect 0.1pg mouse pox virus gene.
Document (is paid auspicious, Yue Bingfei, He Zhengming. the foundation of mouse pox virus PCR detection method and the application in mouse biological product determination. Laboratory Animal Science .2012.29 (3): 12-14.) design pair of primers according to mouse pox virus conserved sequence crmD, establish mouse pox virus PCR detection method.
But specificity and the susceptibility of the current PCR primer for mouse pox virus design still have much room for improvement, and have not yet to see the relevant report of mouse pox virus fluorescent quantitative PCR detection method.
Summary of the invention
The invention provides a kind of fluorescence quantification PCR primer for detecting mouse pox virus to and probe, utilize this primer pair and probe to carry out quantitative fluorescent PCR, can special, detect mouse pox virus delicately.
For the fluorescence quantification PCR primer that detects mouse pox virus to and probe, the base sequence of described primer pair is:
Upstream primer: 5 '-ACAAGGGGTTGGGTGTAAGA-3 ';
Downstream primer: 5 '-GCGTGCTAGTGGTTGCATTA-3 ';
The base sequence of described probe is: 5 '-R-ACCAAACTGGGTAGACGATGT-Q-3 ';
Wherein, R is fluorescent reporter group, and Q is fluorescent quenching group.
The gene order of two strain Ectromelia virus (Ectromelia virusculture-collection ATCC:VR-1431 and Ectromelia virus strain Moscow) full-length genome is downloaded from the FTP of NCBI, have chosen 183 genes and Nt storehouse contrasts from the full-length genome of Ectromelia virus culture-collection ATCC:VR-1431 (JQ410350.1), the ERPV_027 alternatively gene that in therefrom have chosen kind, special and between planting otherness is best, 480 ~ 800 bit sequence sections for ERPV_027 gene devise above-mentioned fluorescence quantification PCR primer to and probe.
In the present invention, described fluorescent reporter group can select FAM, JOE or HEX, is preferably FAM; Described fluorescent quenching group can select TAMRA, Eclipse or BHQ1, is preferably TAMRA.
Present invention also offers a kind of method detecting mouse pox virus, comprise the following steps:
(1) DNA of testing sample is extracted;
(2) with described DNA for template, utilize described fluorescence quantification PCR primer to and probe, carry out real-time fluorescence quantitative PCR;
(3) pcr amplification product is carried out agarose gel electrophoresis separation, dyeing, according to colour developing result, testing sample is identified.
If mouse pox virus content is very low in measuring samples, easily cause corresponding pathogenic bacteria being detected.For avoiding occurring " false negative " result, as preferably, in the reaction system of described real-time fluorescence quantitative PCR, the concentration of template DNA is not less than 21.7 × 10
-8ng/ μ l.21.7 × 10
-8ng/ μ l be namely fluorescence quantification PCR primer of the present invention to the detectability of probe to mouse pox virus.
As preferably, the reaction system of described real-time fluorescence quantitative PCR is: (composition is see TaqMan Fast Advanced Master Mix test kit specification sheets for 2 × TaqMan FastAdvanced Master Mix, purchased from Life Technologies company) 12.5 μ l, the each 0.5 μ l of upstream and downstream primer of 10 μMs, the probe 1.0 μ l of 5 μMs, the template DNA 1.0 μ l of 10ng/ μ l, moisturizing to 25 μ l.
As preferably, the reaction conditions of described real-time fluorescence quantitative PCR is: Real-time PCR optimum reaction condition is: (this temperature is UNG enzyme optimal reaction temperature to 50 DEG C of cultivation 2min, UNG enzyme is included in 2 × TaqMan Fast Advanced Master Mix), 95 DEG C of denaturation 10min; 95 DEG C of sex change 15s, 55 DEG C of annealing 20s, 72 DEG C extend 20s, pre-amplification 10 circulations; 95 DEG C of sex change 15s, 60 DEG C of annealing extend 1min, totally 40 circulations.
Non-specific PCR amplification and pollution can be prevented containing UNG enzyme in reaction system of the present invention, increase the accuracy of PCR result; By increasing in advance and sample low for template concentrations suitably can well being amplified, increase recall rate, reduce limit of detection; Probe is used to carry out fluorescent quantitation detection, high specificity, simple to operate, save time, the object of rapid detection can be reached.
Present invention also offers described fluorescence quantification PCR primer and prepare the application in mouse pox virus identification kit to probe.
Present invention also offers the PCR kit for fluorescence quantitative for detecting mouse pox virus, containing the fluorescence quantification PCR primer for detecting mouse pox virus of the present invention to and probe.
As preferably, also containing positive control dna in described PCR kit for fluorescence quantitative; The base sequence of described positive control dna is as shown in SEQ ID No.1.
Compared with prior art, beneficial effect of the present invention is:
Fluorescence quantification PCR primer of the present invention has extremely strong specificity to probe, only can amplify the specific fragment of ERPV_027 gene in mouse pox virus; And 21.7 × 10 are limited to the detection of mouse pox virus
-8ng/ μ l, substantially increases detection sensitivity.
Accompanying drawing explanation
Fig. 1 carries out electrophoresis result figure when regular-PCR increases to type strain ECT-BJ respectively for utilizing 3 groups of primer pairs (ECT-027, ECT-150, ECT-177);
Wherein, M is 100bp DNA ladder Marker, lower same;
Fig. 2 utilizes ECT-33221 primer pair to carry out electrophoresis result figure when regular-PCR increases to type strain ECT-BJ;
Fig. 3 is the amplification utilizing 4 groups of primer pairs respectively type strain ECT-GZ sample to be carried out to regular-PCR;
Wherein, 1 ~ 4 swimming lane is followed successively by primer ECT-027, ECT-150, ECT-177, ECT-33221 amplification to type strain ECT-GZ;
Fig. 4 utilizes ECT-027, ECT-150, ECT-177 primer pair to carry out electrophoresis result figure when regular-PCR increases to each mouse pox virus DNA sample respectively;
Wherein, 1 ~ 5 swimming lane be followed successively by primer ECT-027 to ECT-BJ, 001, the amplification of HZ, MB35,1436R; 6 ~ 10 swimming lanes be followed successively by primer ECT-150 to ECT-BJ, 001, the amplification of HZ, MB35,1436R; 11 ~ 15 swimming lanes be followed successively by primer ECT-177 to ECT-BJ, 001, the amplification of HZ, MB35,1436R;
Fig. 5 utilizes ECT-33221 primer pair to carry out electrophoresis result figure when regular-PCR increases to each mouse pox virus DNA sample respectively;
Wherein, 1 ~ 5 swimming lane be followed successively by primer ECT-33221 to ECT-BJ, 001, the amplification of HZ, MB35,1436R;
Fig. 6 is the amplification utilizing 4 groups of primer pairs respectively blank to be carried out to regular-PCR;
Wherein, 1 ~ 4 swimming lane is followed successively by primer ECT-027, ECT-150, ECT-177, ECT-33221 amplification to blank;
Fig. 7 be after optimizing reaction system ECT-027 primer pair to the amplification of each mouse pox virus DNA sample;
Wherein, 1 ~ 8 swimming lane be followed successively by primer ECT-027 to negative control (NTC), ECT-GZ-2, ECT-BG, 001, the amplification of MB35,1436R, HZ, ECT-GZ-1;
Fig. 8 A is the Real-time amplification curve that ECT-027 primer pair Taqman fluorescence probe quantitative detects each mouse pox virus DNA sample;
Wherein, Delta Rn represents that standard indicator signal value deducts baseline signal value, and Cycle Number represents PCR cycle number;
Fig. 8 B is the Real-time amplification curve that ECT-027 primer pair Taqman fluorescence probe quantitative detects negative control (NTC);
Fig. 9 A is that ECT-027 primer pair and Taqman probe infect the electrophoresis result figure after the Real-time pcr amplification of sample DNA sample to other viral nucleic acids each and each mouse pox virus;
Wherein, 1 ~ 2 swimming lane is followed successively by a59 virus-MHV, JHM-MHV, and 3 swimming lanes are vaccinia virus, 4 ~ 5 swimming lanes are followed successively by Sendai virus BJ-SV, GZ-SV, 6 swimming lanes are Tyzzer organism, and 7 ~ 16 swimming lanes are followed successively by mouse pox virus and infect No. 1 ~ 10, sample, and 17 swimming lanes are negative control NTC;
Fig. 9 B is that ECT-027 primer pair and Taqman probe carry out the electrophoresis result figure after Real-time pcr amplification to each DNA of bacteria and each mouse pox virus infection sample DNA sample;
Wherein, 18 ~ 24 swimming lanes are followed successively by: Salmonellas, listeria bacteria, segmental bronchus Bao Te bacterium, escherichia coli, Pseudomonas aeruginosa, streptococcus aureus, Candida albicans, 26 ~ 33 swimming lanes are followed successively by mouse pox virus and infect No. 11 ~ 18, sample, and 34 swimming lanes are negative control NTC;
Figure 10 A is ECT-027 primer pair cloned plasmids Taqman probe canonical plotting;
Wherein, Standard Curve represents typical curve;
Figure 10 B is the amplification curve diagram of ECT-027 primer cloned plasmids typical curve;
Figure 11 is the Real-time pcr amplification result figure that ECT-027 primer Taqman probe susceptibility detects to each concentration ERPV_027 cloned plasmids;
Wherein, 1 ~ 9 swimming lane is followed successively by: 21.7 × 10
-9ng/ μ l, 21.7 × 10
-8ng/ μ l, 21.7 × 10
-7ng/ μ l, 21.7 × 10
-6ng/ μ l, 21.7 × 10
-5ng/ μ l, 21.7 × 10
-4ng/ μ l, 21.7 × 10
-3ng/ μ l, 21.7 × 10
-2ng/ μ l, 21.7 × 10
-1ng/ μ l;
Figure 12 A is ECT-027 primer pair and Taqman probe to the Real-time pcr amplification result figure of mouse pox virus type strain and each gerbil jird fecal sample DNA;
Wherein, 1 swimming lane is negative control NTC, and 2 swimming lanes are ECT-BJ, and 3 ~ 7 swimming lanes are followed successively by gerbil jird fecal sample 61 ~ 65;
Figure 12 B is that ECT-027 primer pair and Taqman probe are to the Real-time pcr amplification result figure of each gerbil jird fecal sample DNA;
Wherein, 8 ~ 24 swimming lanes are followed successively by gerbil jird fecal sample 66 ~ 74,76 ~ 83;
The Real-time pcr amplification result figure of Figure 12 C to be ECT-027 primer pair and Taqman probe to each mouse pox virus type strain, mouse pox virus infect sample, gerbil jird liver, gerbil jird lungs sample DNA;
Wherein, 25 ~ 27 swimming lanes are that mouse pox virus infects No. 19 ~ 21, sample, and 28,29 swimming lanes are ECT-GZ-1, ECT-GZ-2, and 30 ~ 35 are followed successively by No. 1 ~ 6, gerbil jird liver samples, and 36 ~ 41 swimming lanes are followed successively by No. 1 ~ 6, gerbil jird lungs sample;
Figure 12 D is that ECT-027 primer pair and Taqman probe are to the Real-time pcr amplification result figure of each gerbil jird ight soil, field rodent fecal sample;
Wherein, 42 ~ 46 swimming lanes are followed successively by No. 84 ~ 87,89, gerbil jird fecal sample, and 47 ~ 49 swimming lanes are followed successively by field rodent fecal sample HN, SH2, SH3.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
One design of primers
183 genes of E.virus culture-collection ATCC:VR-1431 (GenBank Accession No:JQ410350.1) (hereinafter referred to as VR-1431 bacterial strain) and Nt storehouse are contrasted, obtains whole comparison results.
When analyses and comparison result, find that ERPV_027, ERPV_60, ERPV_64, ERPV_77, ERPV_116, ERPV_140, ERPV_142, ERPV_145, ERPV_148, ERPV_149, ERPV_150, ERPV_174, ERPV_175, ERPV_177 gene has the otherness (larger with vaccinia virus difference) between special in extraordinary kind and kind.
Wherein, ERPV_027 (SEQ ID No.1), ERPV_150 (SEQ ID No.2), ERPV_177 (SEQ ID No.3) these three genes all have conservative property in high kind in VR-1431 bacterial strain, E.virus strainMoscow (GenBank Accession No:AF012825.2), E.virus isolate NAVAL (GenBank Accession No:KJ563295.1); And there is larger sequence difference in ERPV_027 gene between VR-1431 bacterial strain and vaccinia virus Cowpox virus strain Norway_1994_MAN (GenBank Accession No:HQ420899.1), there is larger sequence difference in ERPV_150 gene, ERPV_177 gene exists larger sequence difference between VR-1431 bacterial strain and gerbil jird poxvirus Taterapox virusstrain Dahomey (GenBank Accession No:DQ437594.1) between VR-1431 bacterial strain and vaccinia virus Vaccinia virus strainDryvax clone DPP21 (GenBank Accession No:JN654986.1).
Therefore, the present embodiment chooses 480 ~ 800 bit sequence sections, 60 ~ 630 bit sequence sections of ERPV_150 gene, the 1000-1680 bit sequence section of ERPV_177 gene of ERPV_027 gene respectively, as design of primers site.
Simultaneously, the present embodiment is to E.virus strain ECT-33221 (GenBank Accession No:AY102991.1), E.virus strain ECT-K1 (GenBank Accession No:AY102990.1), E.virus strain ECT-4908 (GenBank Accession No:AY102989.1), E.virusstrain ECT-4908 (GenBank Accession No:AY102988.1), after the gene (SEQ ID No.4) of the secreted chemokin e bindingprotein of E.virus strain MP-1 (GenBank Accession No:AY102987.1) carries out sequence alignment, for its 202 ~ 346 bit sequence section as design of primers site.
The fluorescence quantification PCR primer utilizing Primer Express 3.0 to design for above four genes to probe sequence (5 ' end band of probe sequence has fluorescent reporter group FAM, and 3 ' end band has fluorescent quenching group TAMRA) as table 1.
Table 1 four groups of fluorescence quantification PCR primers to Taqman probe sequence
The specific detection of two primers
(1) extract the genomic dna of type strain ECT-BJ (deriving from National Institute for Food and Drugs Control), ECT-GZ (deriving from Guangdong laboratory animal monitoring institute) respectively, DNA is with reference to the extracting of QIAampDNA Mini Kit specification sheets).
1. with type strain ECT-BJ genomic dna for template, utilize ECT-027 primer pair, ECT150 primer pair, ECT-177 primer pair, ECT-33221 primer pair to carry out regular-PCR amplification (only use primer pair, do not use probe) respectively;
Amplification system is: 10 × Taq PCR Buffer is (containing Mg
2+) 2 μ l, dNTPs Mixture (2.5mM) 2 μ l, Taq archaeal dna polymerase (5U/ μ l) 0.5 μ l, each 0.5 μ l of upstream and downstream primer (10 μMs), template DNA (10ng/ μ l) 2 μ l, moisturizing to 20 μ l.
Amplification program is: 95 DEG C of denaturation 5min; 95 DEG C of sex change 15s, 60 DEG C of annealing 20s, 72 DEG C extend 20s, totally 40 circulations; 72 DEG C extend 7min again; 8 DEG C of preservations.
Carry out 2% agarose gel electrophoresis to amplified production, electrophoresis result is shown in Fig. 1 and Fig. 2.From Fig. 1 and Fig. 2, the amplified fragments size of four pairs of primers all meets expection.2. with type strain ECT-GZ genomic dna for template, utilize ECT-027 primer pair, ECT150 primer pair, ECT-177 primer pair, ECT-33221 primer pair to carry out regular-PCR amplification (only use primer pair, do not use probe) respectively;
Amplification system is: 10 × Taq PCR Buffer is (containing Mg
2+) 2 μ l, dNTPs Mixture (2.5mM) 2 μ l, Taq archaeal dna polymerase (5U/ μ l) 0.5 μ l, each 0.5 μ l of upstream and downstream primer (10 μMs), template DNA (10ng/ μ l) 2 μ l, moisturizing to 20 μ l.
Amplification program is: 95 DEG C of denaturation 5min; 95 DEG C of sex change 15s, 60 DEG C of annealing 20s, 72 DEG C extend 20s, totally 40 circulations; 72 DEG C extend 7min again; 8 DEG C of preservations.
Carry out 2% agarose gel electrophoresis to amplified production, electrophoresis result is shown in Fig. 3.As seen from Figure 3, the amplified fragments size of four pairs of primers all meets expection.Each pcr amplified fragment is checked order, by comparing with all mouse pox virus sequence (sequence information is see the present embodiment first part " design of primers ") in Genbank, to determine the accuracy that PCR detects; Result meets expection.
(2) with the mouse pox virus DNA sample of different sources for template, the specificity of four pairs of primers is verified.
For examination mouse pox virus DNA sample be respectively: ECT-BJ, 001, MB35,1436R and HZ (all deriving from National Institute for Food and Drugs Control above), ECT-GZ-1 and ECT-GZ-2 (being the different batches DNA of extracting from ECT-GZ respectively).
1. regular-PCR checking
With reference to QIAamp DNA Mini Kit specification sheets, extract respectively mouse pox virus sample ECT-BJ, 001, the genomic dna of MB35,1436R, HZ, ECT-GZ-1, ECT-GZ-2, utilize four pairs of primers in table 1 to carry out regular-PCR amplification respectively, amplification system as above.Amplification is shown in Fig. 4, Fig. 5, Fig. 6.
As seen from Figure 4, ECT-027 primer pair can increase obtain object band to type strain ECT-BJ, ECT-GZ-1, ECT-GZ-2, strain isolated 001, MB35,1436R, HZ, without amplification in negative control (replacing template with water) (see Fig. 6).
And from Fig. 4, Fig. 5, ECT-150 primer pair, ECT-177 primer pair, ECT-33221 primer pair are all better to the expanding effect of type strain, but strain isolated HZ can not be detected effectively; Without amplification in negative control (replacing template with water) (see Fig. 6).
Show that the specificity of ECT-150 primer pair, ECT-177 primer pair, ECT-33221 primer pair is poor, therefore the present embodiment selects ECT-027 primer to carry out the detection of Taqman fluorescence probe quantitative PCR, and optimization (PCR reaction system is constant) has been done to the PCR response procedures of ECT-027 primer, the PCR response procedures after optimization is:
95 DEG C of denaturation 5min; 95 DEG C of sex change 15s, 55 DEG C of annealing 20s, 72 DEG C extend 20s, totally 40 circulations; 72 DEG C extend 7min again; 8 DEG C of preservations.
Again carry out PCR to each mouse pox virus sample with the reaction system after optimizing, amplification is shown in Fig. 7.Can draw from Fig. 7: ECT-027 primer pair strain isolated HZ can detect effectively, and in type strain, goal gene content is very high, and in strain isolated, content is lower.Do not detect in negative control (NTC).
2. use the above-mentioned mouse pox virus DNA sample of ECT-027 primer pair and negative control (NTC) to carry out TaqMan probe Real-time PCR to detect:
Amplification system is: 2 × TaqMan Fast Advanced Master Mix (composition is see test kit specification sheets) 12.5 μ l, the each 0.5 μ l of upstream and downstream primer (10 μMs), probe (5 μMs) 1.0 μ l, template DNA (10ng/ μ l) 1.0 μ l, moisturizing to 25 μ l;
Amplification program is: cultivate 2min (add UNG enzyme in test kit, this temperature is its optimal reaction temperature), 95 DEG C of denaturation 10min for 50 DEG C; 95 DEG C of sex change 15s, 55 DEG C of annealing 20s, 72 DEG C extend 20s, pre-amplification 10 circulations; 95 DEG C of sex change 15s, 60 DEG C of annealing extend 1min, totally 40 circulations, collect fluorescent signal and reading of data.
Detected result is in table 2, Fig. 8 A, Fig. 8 B.
Table 2ECT-027 is to the Real-time PCR detected result of each mouse pox virus DNA sample
Note: " N/A " expression does not detect numerical value, "-" represents blank.
All have good expanding effect as can be seen from table 2, Fig. 8 A, Fig. 8 B, ECT-027 to type strain and strain isolated, and in type strain DNA, goal gene content is higher, in strain isolated DNA, goal gene content is lower; The amplification curve of ECT-027 to negative control (NTC) do not detected.
3. use ECT-027 primer pair and Taqman probe to carry out regular-PCR amplification to other DNA samples (comprising the DNA of DNA of bacteria, other viral DNAs, mouse pox virus infection sample), detect its specificity.
Extract Salmonellas (Salmonella spp.) respectively, listeria bacteria (Listeriamonocytogenes), segmental bronchus Bao Te bacterium (Bordetella bronchiseptica), escherichia coli (Escherichia coli), Pseudomonas aeruginosa (Pseudomonas aeruginosa), streptococcus aureus (Staphylococcus aureus), the genomic dna (DNA extraction method is the same) of Candida albicans (Monilia albican) (above bacterial strain is Zhejiang Academy of Medical Sciences and preserves),
Extract the genomic dna (DNA extraction method is the same) that mouse pox virus infects sample (the BHK21 cell DNA that mouse pox virus infects) respectively;
Extract the genomic dna (DNA extraction method is the same) of vaccinia virus (cowpox Virus), Tyzzer organism (Tyzzer ' s organism), (all deriving from National Institute for Food and Drugs Control above) respectively;
Extract Mouse hepatitis virus (Mouse Hepatitis Virus) A59-MHV, JHM-MHV, Sendai virus (Sendai Virus) GZ-SV (all deriving from Guangdong laboratory animal monitoring institute above), Sendai virus BJ-SV (deriving from National Institute for Food and Drugs Control) total serum IgE respectively, and it is for subsequent use to obtain cDNA through reverse transcription.
Wherein, the reaction system of reverse transcription is:
Reaction conditions is: 42 DEG C, 1h; 70 DEG C, 15min; 8 DEG C, forever.
Respectively with the genomic dna of said extracted for template, utilize ECT-027 primer to carry out Taqman probe Real-time PCR and detect, quantitative PCR detection the results are shown in Figure 9A, Fig. 9 B.
From each DNA of bacteria, respectively other viral DNAs, all do not amplify target fragment from Fig. 9 A, Fig. 9 B, ECT-027 primer Taqman probe method, infect sample DNA then amplifying target fragment from each mouse pox virus.Show that ECT-027 primer pair and Taqman probe method have good specificity, can increase mouse pox virus specifically.
The sensitivity Detection of three-primer
ERPV_027 cloned plasmids (21.7ng/ μ l) is done successively 10 times of dilutions until 21.7 × 10
-9ng/ μ l, the ERPV_027 cloned plasmids getting each extension rate of 2 μ l again, as template, utilizes ECT-027 primer pair and Taqman probe to carry out Real-time PCR detection, PCR amplification system and response procedures the same, as shown in Figure 10, electrophoresis result is shown in Figure 11 to amplification curve.
As seen from Figure 10, in 40 PCR circulation, minimumly can detect 21.7 × 10
-8ng/ μ l, 21.7 × 10
-9ng/ μ l can't see complete amplification curve.As seen from Figure 11, along with template concentrations increases, target stripe brightness brightens successively, and template concentrations is 21.7 × 10
-9obvious band is can't see during ng/ μ l.Show that the detection of ECT-027 to mouse pox virus ERPV_027 is limited to 21.7 × 10
-8ng/ μ l.
Four stability and repeated experiment
Get DNA and with a collection of PCR detection reagent when-20 DEG C of refrigerators place 1 week, 2 weeks, 3 weeks, ECT-027 primer pair and Taqman probe is utilized to carry out Real-time PCR detection, 3 repetitions are respectively done in 3 tests, PCR amplification system and response procedures the same, amplification is in table 3.
Table 3ECT-027 is to the Real-time PCR detected result of each mouse pox virus DNA sample
From table 3, the RT-PCR method stability utilizing ECT-027 primer pair and Taqman probe to set up and repeatability all better.
Five actual detections are applied
Using the DNA of type strain ECT-BJ, ECT-GZ-1, ECT-GZ-2 as positive control, with ECT-027 primer Taqman probe method respectively to take from mouse animal excrement, tissue etc. 42 increment this DNA sample and the mouse pox virus DNA sample that infects sample (mouse pox virus infect BHK21 cell DNA) carry out Real-time PCR detection, the electrophoresis result of PCR primer is shown in Figure 12 A, Figure 12 B, Figure 12 C, Figure 12 D.
From Figure 12 A, Figure 12 B, Figure 12 C, Figure 12 D, ECT-027 primer Taqman probe method does not all detect mouse pox virus goal gene from the DNA of mouse animal excrement, tissue samples, but infects the DNA of sample from mousepox and goal gene detected and content is higher.
Claims (9)
1. for the fluorescence quantification PCR primer that detects mouse pox virus to and probe, it is characterized in that, the base sequence of described primer pair is:
Upstream primer: 5 '-ACAAGGGGTTGGGTGTAAGA-3 ';
Downstream primer: 5 '-GCGTGCTAGTGGTTGCATTA-3 ';
The base sequence of described probe is: 5 '-R-ACCAAACTGGGTAGACGATGT-Q-3 ';
Wherein, R is fluorescent reporter group, and Q is fluorescent quenching group.
2. fluorescence quantification PCR primer as claimed in claim 1 is preparing the application in mouse pox virus detection kit to probe.
3., for detecting the PCR kit for fluorescence quantitative of mouse pox virus, it is characterized in that, containing, for example the fluorescence quantification PCR primer for detecting mouse pox virus according to claim 1 to and probe.
4. PCR kit for fluorescence quantitative as claimed in claim 3, is characterized in that, containing positive control dna.
5. PCR kit for fluorescence quantitative as claimed in claim 4, it is characterized in that, the base sequence of described positive control dna is as shown in SEQ ID No.1.
6. detect a fluorescence quantifying PCR method for mouse pox virus, it is characterized in that, comprise the following steps:
(1) DNA of testing sample is extracted;
(2) with described DNA for template, utilize fluorescence quantification PCR primer as claimed in claim 1 to and probe, carry out real-time fluorescence quantitative PCR;
(3) pcr amplification product is carried out agarose gel electrophoresis separation, dyeing, according to colour developing result, testing sample is identified.
7. fluorescence quantifying PCR method as claimed in claim 6, it is characterized in that, in the reaction system of described real-time fluorescence quantitative PCR, the concentration of template DNA is not less than 21.7 × 10
-8ng/ μ l.
8. fluorescence quantifying PCR method as claimed in claims 6 or 7, it is characterized in that, the reaction system of described real-time fluorescence quantitative PCR is: 2 × TaqMan Fast Advanced MasterMix12.5 μ l, the each 0.5 μ l of upstream and downstream primer of 10 μMs, the probe 1.0 μ l of 5 μMs, the template DNA 1.0 μ l of 10ng/ μ l, moisturizing to 25 μ l.
9. fluorescence quantifying PCR method as claimed in claim 8, it is characterized in that, the reaction conditions of described real-time fluorescence quantitative PCR is: cultivate 2min, 95 DEG C of denaturation 10min for 50 DEG C; 95 DEG C of sex change 15s, 55 DEG C of annealing 20s, 72 DEG C extend 20s, pre-amplification 10 circulations; 95 DEG C of sex change 15s, 60 DEG C of annealing extend 1min, totally 40 circulations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410734124.2A CN104450964A (en) | 2014-12-05 | 2014-12-05 | Fluorescent quantitative polymerase chain reaction (PCR) method for detecting mouse poxvirus as well as special primer pair, probe and kit of method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410734124.2A CN104450964A (en) | 2014-12-05 | 2014-12-05 | Fluorescent quantitative polymerase chain reaction (PCR) method for detecting mouse poxvirus as well as special primer pair, probe and kit of method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104450964A true CN104450964A (en) | 2015-03-25 |
Family
ID=52897709
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410734124.2A Pending CN104450964A (en) | 2014-12-05 | 2014-12-05 | Fluorescent quantitative polymerase chain reaction (PCR) method for detecting mouse poxvirus as well as special primer pair, probe and kit of method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104450964A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111850099A (en) * | 2019-04-25 | 2020-10-30 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | RPA kit, primers, probe and method for detecting varicella virus |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102329889A (en) * | 2011-08-16 | 2012-01-25 | 舒泰神（北京）生物制药股份有限公司 | Primer and probe and method for detecting poxvirus muris |
| CN102399907A (en) * | 2011-11-25 | 2012-04-04 | 舒泰神（北京）生物制药股份有限公司 | Multiplex real-time fluorescent polymerase chain reaction (PCR) primer for simultaneously detecting mouse adenovirus and poxvirus muris mouse pox virus, probe as well as reagent kit thereof |
-
2014
- 2014-12-05 CN CN201410734124.2A patent/CN104450964A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102329889A (en) * | 2011-08-16 | 2012-01-25 | 舒泰神（北京）生物制药股份有限公司 | Primer and probe and method for detecting poxvirus muris |
| CN102399907A (en) * | 2011-11-25 | 2012-04-04 | 舒泰神（北京）生物制药股份有限公司 | Multiplex real-time fluorescent polymerase chain reaction (PCR) primer for simultaneously detecting mouse adenovirus and poxvirus muris mouse pox virus, probe as well as reagent kit thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111850099A (en) * | 2019-04-25 | 2020-10-30 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | RPA kit, primers, probe and method for detecting varicella virus |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Rodrigues et al. | Molecular diagnosis of pathogenic Sporothrix species | |
| CN111074000B (en) | Triple fluorescence quantitative PCR detection material and kit for distinguishing ASFV wild strain from double gene deletion strain | |
| CN107988325B (en) | RAA constant temperature fluorescence detection method and reagent for shrimp liver Enterocytozoon (EHP) | |
| CN107988428A (en) | Shrimp irido virus（SIV）RAA constant temperature fluorescence detection method and reagent | |
| CN107988326A (en) | Prawn Acute Hepatic pancreatic necrosis（AHPND）RAA constant temperature fluorescence detection method and reagent | |
| CN107988427A (en) | Prawn hepatopancreatic parvovirus（HPV）RAA constant temperature fluorescence detection method and reagent | |
| Soltany-Rezaee-Rad et al. | Comparison of SYBR Green and TaqMan real-time PCR methods for quantitative detection of residual CHO host-cell DNA in biopharmaceuticals | |
| CN107988426A (en) | Prawn Taura syndrome（TSV）RAA constant temperature fluorescence detection method and reagent | |
| CN103333903A (en) | Target sequence, primer and probe for detecting helicobacter pylori and kit thereof | |
| Liu et al. | Development of cross-priming amplification coupled with vertical flow visualization for rapid detection of infectious spleen and kidney necrosis virus (ISKNV) in mandarin fish, Siniperca chuatsi | |
| CN107828914A (en) | Infectious subcutaneous and haematopoietic necrosis virus（IHHNV）RAA constant temperature fluorescence detection method and reagent | |
| Zeng et al. | Molecular detection of genotype II grass carp reovirus based on nucleic acid sequence-based amplification combined with enzyme-linked immunosorbent assay (NASBA-ELISA) | |
| Dong et al. | Development and evaluation of a droplet digital PCR assay for the detection of fowl adenovirus serotypes 4 and 10 in attenuated vaccines | |
| CN105200162A (en) | HRM detection method for rapidly distinguishing HP-PRRS live vaccine JXA1-R strains and wild strains and primer of HRM detection method | |
| CN102634605B (en) | Method for detecting egg drop syndrome viruses and kit for method | |
| Woźniakowski et al. | The development and evaluation of cross‐priming amplification for the detection of avian reovirus | |
| CN109439801A (en) | A kind of honeybee Israel acute paralysis virus real-time fluorescent RT-PCR detection reagent box and its detection method | |
| Zhang et al. | Establishment and application of real-time fluorescence-based quantitative PCR for detection of infectious laryngotracheitis virus using SYBR Green I | |
| CN104195265A (en) | PCR-HRM primer and method for rapidly distinguishing wild strain and vaccine strain of canine parvovirus | |
| CN104450964A (en) | Fluorescent quantitative polymerase chain reaction (PCR) method for detecting mouse poxvirus as well as special primer pair, probe and kit of method | |
| CN103397108A (en) | HCLV (hog cholera lapinized virus) fluorescent quantitation RT-PCR (reverse transcription-polymerase chain reaction) detection kit | |
| CN104498593A (en) | Primer pair and kit for identification or assisted identification of stored bean weevils | |
| CN105200122A (en) | Quantitative detection kit for wheat stripe rust and application thereof | |
| CN113046481B (en) | Primer, probe and kit for quantitative fluorescence detection of pigeon adenovirus B | |
| CN105861751B (en) | A kind of primer pair for detecting mouse adenovirus, PCR kit for fluorescence quantitative and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150325 |