CN102618665B - Kit for fluorescence PCR detection of herpes simplex virus I - Google Patents
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Abstract
The invention relates to a kit for fluorescence PCR detection of a herpes simplex virus I, and especially relates to a kit for rapid and qualitative detection of the herpes simplex virus I infection through a real-time fluorescence polymerase chain reaction technology. The kit has a very high sensitivity and specificity, makes the qualitative detection of I nucleic acid DNA in a sample be realized, and is suitable for the diagnosis of the herpes simplex virus I infection, curative effect monitoring of the antiviral therapy, and epidemiological researches.
Description
Technical field
The present invention relates to the test kit that a kind of fluorescent PCR detects herpes simplex virus I-type, particularly relate to the test kit quick with real-time fluorescence polymerase chain reaction technique, qualitative detection herpes simplex virus I-type infects.This test kit has very high sensitivity and specificity, realize herpes simplex virus I-type nucleic acid DNA in sample is carried out to qualitative detection by test kit of the present invention, be applicable to the auxiliary diagnosis that herpes simplex virus I-type infects, the curative effect monitoring of antiviral therapy and epidemiological study.
Background technology
Hsv herpes simplex virus, belongs to herpetoviridae a virus subfamily, virus particle size approximately 180 nanometers.At present this virus is divided into 1 type and 2 types according to antigenic difference.1 type is mainly obtained by lip focus, and 2 types can be separated to from sexual organ focus.Infection is the contact due to person to person.Within four months from occurring, can reach the 50-90% of population to infected number of several years, be the one virus of the most easily invading people, but in clinical only some morbidity.This disease can be divided into: lip bleb, herpetic keratitis, herpetic dermatitis, Herpes genitalis, kaposi's disease etc. are also the cause of disease of meningitis, encephalitis sometimes.Lip portion bleb is more easily diagnosis generally, simultaneously because all stimuluss such as Exposure to Sunlight, heating cause recurrence.This virus can be on chick chorioallantoic membrane and people's, monkey, chicken etc. animal culturing cell in propagation in large quantities.
HSV has typical simplexvirus morphological specificity.Be divided into two serotypes, i.e. HSV-1 and HSV-2 according to biological chemistry, biology, epidemiology.The two genome is similar, and sequence has 50% homology, the about 152kb of HSV genome, 34 genes, more than 70 polypeptide (polypeptides) of encoding.Particularly in the late protein of genes encoding, have 11 kinds of envelope glycoproteins (gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL, gM), some function is clearer.Wherein gB and gD and viruses adsorption and penetrate is relevantly the viral ligand molecular with cell-specific acceptor interaction.The ability that gD induction produces neutralizing antibody is the strongest, can be used for developing vaccine.GC is that complement C3b-is in conjunction with albumen (complement C3b-binding protein).GE is Fc acceptor, can hold combination with the Fc of IgG.GG is type specific antigen, can distinguish HSV-1 (gG-1) and HSV-2 (gG-2) with this antigen.GH is relevant with viral release.
HSV is wider to zoogenetic infection host range.Common experimental animal is rabbit, cavy and mouse etc.HSV can breed in various kinds of cell, the passage cell culture of isolated viruses such as conventional primary rabbit kidney, HEKC and suslik kidney.There is obvious cytopathy in cells infected, and occurs acidophilic intranuclear inclusion very soon.Hsv extensively distributes in the whole world, in crowd infect very general, hide and recurrent infection person more.Patient and carrier are this sick contagium.Virus can enter body by the direct contact of skin, mucous membrane or property route of exposure.
Newborn infant's bleb is common and serious clinically infection, and mortality ratio exceedes 50% according to statistics, and survivor approximately has 1/2 major injury.HSV-1, HSV-2 all can infect newborn infant by birth canal in the time of childbirth, taking HSV-2 as common, account for 75%.Often occur in raw latter the 6th day.Infection type has: the 1. local damage in skin, eye and oral cavity; 2. encephalitis; 3. to internal organ, there is pyemia (sepsis) in viral spread, often causes death.The early stage anti-infective mortality ratio that reduces.C-section is the effective ways of avoiding genital tract infection.Gravid woman infects HSV-1, and virus is diaplacental infection fetus likely, causes miscarriage, stillborn foetus or congenital abnormality.
herpes simplex virus I-typecause the infection of more than waist skin, eye, herpes of mouth and organ (brain), the primary infection of HSV-1 is often confined to pars oralis pharyngis more, especially common with gingivostomatitis (gingivostomatitis).Can cause in addition encephalitis, skin bleb eczema.Adult can cause pharyngitis and tonsillitis.Discovered in recent years HSV-1 is also one of major reason causing genital herpes.
Microbiological examination method comprises:
Virus separation and Culture is the reliable basis of the current herpesvirus infection of clarifying a diagnosis clinically.Can gather the samples such as the blister liquid, cerebrospinal fluid, cornea scraping thing, saliva of the diseased region such as skin, sexual organ, inoculation human diploid fibroblasts strain WI38 and other passage cell strain are as Vero, BHK etc., after 24～48 hours, there is swelling, become the pathologies such as circle, cytogamy in cell.Then do immunofluorescence dyeing qualification or apply DNA analysis of Restriction Endonuclease Profile and shape by the monoclonal antibody of HSV-1 and HSV-2.
Antibody test: the method that is usually used in antibody test has complement fixation test (CFT), neutralization test, immunofluorescence and enzyme linked immunosorbent assay etc., the clinical detection that is used for acute infection diagnosis and Organ Transplantation Patients, and epidemiology survey.As for acute infection diagnosis, should take acute phase and decubation paired sera, detect IgG and IgM in serum simultaneously.
DNA detection: get pathological tissues or cell, extract viral DNA, hybridize with the HSV DNA probe of mark or the gB glycoprotein gene of apply PCR detection HSV-1 or HSV-2 judges whether it is the infection of HSV.This method is for doubting the diagnosis for HSV Patients With Encephalitis.
And real-time fluorescence PCR technology is a kind of new PCR method, be on the basis of conventional PCR reaction, increased can with the probe of amplification template specific binding, compared with normal PCR, the method is without PCR product is processed again, be the whole process that completes detection under closed state completely, there is the features such as real-time, accurate, quick.
This test kit adopts TaqMan fluorescent probe technique, the special special conserved regions of height of choosing herpes simplex virus I-type gene coding region is target region, design Auele Specific Primer and fluorescent probe, wherein object fluorescent probe 5 ' end mark fluorescent transmitting group FAM, the non-fluorescent quenching group B of 3 ' end mark HQ1, utilizes fluorescent PCR detector to detect fluorescent signal at FAM passage.Be provided with interior mark quality control system, for the quality control of whole nucleic acid extraction and reaction system simultaneously.Interior target area is chosen consistent with HSV-1 target area, and at probe location synthetic one section of oligonucleotide, design specificity fluorescent probe, with the equal no cross reaction of any species, wherein fluorescent probe 5 ' end mark fluorescent transmitting group HEX, and mark system of quality control in forming with interior mark plasmid is common, can detect fluorescence at VIC passage.For thing to be checked, if detected result shows typical S type fluorescence growth curve, positive sample, on the contrary negative.By nucleic acid extraction system simple to operation, in conjunction with real-time fluorescence PCR detection system operation pcr amplification program, this test kit has been realized the high-level efficiency detecting, highly sensitive and high special.
Summary of the invention
The present invention relates to the test kit that a kind of fluorescent PCR detects herpes simplex virus I-type, particularly relate to the test kit quick with real-time fluorescence polymerase chain reaction (RT-PCR) technology, qualitative detection herpes simplex virus I-type infects.The method of application fluorescent PCR, carries out qualitative detection to the herpes simplex virus I-type DNA in bleb liquid, urogenital tract secretion equal samples, can be used for the auxiliary diagnosis that herpes simplex virus I-type infects, the curative effect monitoring of antiviral therapy and epidemiological study.Its ultimate principle is to utilize real-time fluorescence PCR technology, and taking the special special conserved regions of the height of herpes simplex virus I-type gene coding region as target region, design Auele Specific Primer and fluorescent probe, carry out pcr amplification, for the qualitative detection of herpes simplex virus I-type DNA.In addition, this test kit, also with internal standard substance matter, is monitored for the whole process to nucleic acid extraction, reduces the appearance of false negative result.
In order to realize the present invention, we have adopted following technical scheme:
1) use suitable foranalysis of nucleic acids software respectively the nucleotide sequence of known herpes simplex virus I-type gene to be carried out to homology comparison, on the basis of finding out homology segment, further use suitable primer-design software to select and design oligonucleotides primer and probe.Because designed primer and probe all have the sequence that is complementary to HSV-1 gene, and there is no homology with the nucleotide sequence of other pathogenic agent, do not comprise the restriction enzyme site of any common endonuclease yet, so false negative and the false positive of having avoided herpes simplex virus I-type to detect, improved the reliability and the accuracy that detect.Be provided with interior mark quality control system, for the quality control of whole reaction system simultaneously.Interior target area is chosen consistent with HSV-1 target area, and at probe location synthetic one section of oligonucleotide, design specificity fluorescent probe, with the equal no cross reaction of any species.
2) use synthetic required Oligonucleolide primers and the probe of DNA synthesizer, with carrying out the processing of ammonia solution after molecular sieve and fast protein liquid chromatography method (FPLC) purifying.Same synthetic required probe sequence, ammonia solution after processing respectively at its 5 ' end mark as 6-FAM amidite and the HEX of fluorescence generation group (reporter group), and its 3 ' end mark by active connecting arm coupling on as the BHQ1 of fluorescent quenching or inhibitor group.With the fluorescently-labeled probe of FPLC purification by chromatography.Then, polyacrylamide gel (20%) electrophoretic method under use Denaturing and primer and the probe of spectrophotometry physical characterization synthesized.
3) be suitable for the reaction system of pcr amplification.Reaction system comprises warm start Taq enzyme, 2 '-deoxynucleoside triphosphate, can with the forward primer of the Article 1 chain combination of double-stranded target polynucleotide, can with the reverse primer of the Article 2 chain combination of double-stranded target polynucleotide, can be combined with target polynucleotide and two ends are combined with respectively the oligonucleotide probe of fluorescence generation group and fluorescent quenching group, can be combined with interior mark Nucleotide and two ends are combined with respectively target oligonucleotide probe in the detection of fluorescence generation group and fluorescent quenching group, the damping fluid that contains magnesium ion.Be provided with interior mark quality control system, for the quality control of whole nucleic acid extraction and reaction system simultaneously.
4) from sample to be tested, extract nucleic acid DNA, add respectively in aforesaid reaction system, directly through pcr amplification, from amplified fluorescence curve, the nucleic acid DNA of unknown sample is carried out to qualitative detection.
Test kit based on above technical scheme invention comprises: (1) nucleic acid extracting reagent, HSV-1 reaction solution A, HSV-1 reaction solution B, HSV-1 inner mark solution, negative quality control product, HSV-1 strong positive quality control product, the critical positive quality control product of HSV-1, and the packing box of these reagent bottles of packaging or pipe is separated and concentrated in (2).
According to a preferred embodiment of the invention, wherein PCR reaction system comprises HSV-1 reaction solution A and HSV-1 reaction solution B.
According to another preferred embodiment of the present invention, wherein HSV-1 reaction solution A (primer probe mixed solution) is made up of forward primer, reverse primer, object oligonucleotide probe, interior mark oligonucleotide probe and PCR reaction buffer, it is characterized in that for forward and the reverse primer of target polynucleotide amplification be respectively 5 '-TTATCCCATTCCTTTTGGTTCTT-3 ' (SEQ ID NO:1) and 5 '-GGGTCATGTTGGGGGCTT-3 ' (SEQ ID NO:2).
According to another preferred embodiment of the present invention, be further characterized in that for the oligonucleotide probe of target polynucleotide amplification be 5 '-X-TGGGGTTGGGTGGTGGAGGAGA-Y-3 ' (SEQ ID NO:3), wherein X/Y represents fluorescent mark group, comprises and can be combined with target polynucleotide and the two ends oligonucleotide probe that has fluorescence generation group and fluorescent quenching group of combination respectively.
According to another preferred embodiment of the present invention, be further characterized in that for the primer concentration and probe concentration of target polynucleotide amplification and be 5～15pmol/ person-portion, preferably primer concentration is that 10pmol/ person-portion, concentration and probe concentration are 5pmol/ person-portion.
According to another preferred embodiment of the present invention, in primer probe mixed solution, be 5 '-X-CCTGCTCTGACTCTGACCATCCCTG-Y-3 ' (SEQ ID NO:4) for the oligonucleotide probe of interior mark polynucleotide amplification, wherein X/Y represents fluorescent mark group, comprises and can be combined with interior mark polynucleotide and the two ends oligonucleotide probe that has fluorescence generation group and fluorescent quenching group of combination respectively.
According to another preferred embodiment of the present invention, in primer probe mixed solution, be wherein 3～10pmol/ person-portion for the oligonucleotide probe concentration of interior mark polynucleotide amplification, preferably concentration and probe concentration is 5pmol/ person-portion.
According to another preferred embodiment of the present invention, HSV-1 reaction solution B is made up of warm start Taq enzyme, dNTPs, enzyme diluent.
According to another preferred embodiment of the present invention, be further characterized in that warm start Taq enzyme dosage is that 3～7U/ person-portion, dNTPs concentration are 0.20mmol/L, enzyme diluent is general commercially available enzyme diluent.
According to another preferred embodiment of the present invention, HSV-1 reaction solution A comprises PCR reaction buffer, and wherein PCR reaction buffer comprises 10mmol/L Tris-HCl (pH8.8), 50mmol/L KCl, 3.0mmol/L MgCl
2.
According to a preferred embodiment of the invention, negative quality control product is physiological saline, and the critical positive quality control product of HSV-1 strong positive quality control product and HSV-1 is and contains HSV-1 virus strain, and concentration is respectively 5.0 × 10
6with 5.0 × 10
3iU/ml.
According to another preferred embodiment of the present invention, for optimal reaction temperature and the time of pcr amplification be wherein: 95 DEG C of 15min, 1 circulation; Last 94 DEG C of 15s, 55 DEG C of 45s, 40 circulating collection fluorescent signals.
According to a preferred embodiment of the invention, wherein the sample to be checked of test kit is bleb liquid, urogenital tract secretion, reproductive tract scraping blade, cerebrospinal fluid, tracheal bronchus secretory product, bronchoalveolar lavage fluid etc.
The sensitivity that PCR test kit provided by the invention can detect herpes simplex virus I-type is 100copies/ml, illustrates that this test kit has extraordinary sensitivity.
The present invention is directed to herpes simplex virus I-type gene design Auele Specific Primer and probe, the linearity range of detection is 2.5 × 10
2copies/ml-1.0 × 10
8copies/ml, with same kind, infection site is identical or infection symptoms is similar other viruses (herpes simplex virus type II, human cytomegalic inclusion disease virus, Epstein-Barr virus etc.) no cross reaction.
The present invention compared with prior art, has the following advantages: (advantage after the technology of existing detection herpes simplex virus I-type and the contrast of this technology)
(1) totally-enclosed reaction, after extracting DNA, is directly used in PCR and detects, and has avoided polluting occurring.
(2) test kit, with internal standard substance matter, can be monitored for the whole process to nucleic acid extraction and pcr amplification, thereby reduces the appearance of false negative result.
(3) use warm start Taq enzyme, prevent low temperature non-specific amplification, the sensitivity of test kit is improved.
(4) relatively traditional cultural method, can rapid detection sample amplifying nucleic acid DNA by real time fluorescent PCR method.
Brief description of the drawings
Fig. 1 shows the detected result of HSV-1 strong positive quality control product, the critical positive quality control product of HSV-1 and negative quality control product.HSV-1 strong positive quality control product, the critical positive quality control product FAM detection path of HSV-1 amplification curve have obvious increased logarithmic phase, be typical S type amplification curve, and the definite value of strong positive and critical positive quality control product are respectively 1.0 × 10
6-5.0 × 10
7copies/ml and 5.0 × 10
2-5.0 × 10
4within the scope of copies/ml, can clearly be judged to be the positive.Negative quality control product FAM detection path amplification curve is without increased logarithmic phase, and VIC detection path amplification curve is increased logarithmic phase; Can clearly be judged to be feminine gender, all meet quality control judging criterion.
Fig. 2 shows the specific detected result of HSV-1 RNA.The amplification curve of 10 parts of specificity reference materials is straight or oblique lower and with baseline without intersecting, there is no Ct value, can clearly be judged to be feminine gender.Specificity sample comprises herpes simplex virus type 2 (HSV-2), varicella zoster virus (VZV), human cytomegalic inclusion disease virus (CMV), other pathogenic micro-organism samples such as Epstein-Barr virus.
The detected result of Fig. 3 display line personality sensitivity reference material.Totally 6 parts of linear sensitivity reference materials, label is respectively L1-L6, demarcates its copy number by measuring 0D value after HSV-1 virus liquid extraction nucleic acid, and the HSV-1 virus liquid of having demarcated is diluted to 1 × 10 successively
7copies/ml, 1 × 10
6copies/ml, 1 × 10
5copies/ml, 1 × 10
4copies/ml, 1 × 10
3copies/ml, 2.5 × 10
2copies/ml, as linear reference product, numbers L1-L6.L1-L6 taking preparation carries out duplicate detection 2 times as sample to be checked.
Fig. 4 shows the detected result of 10 parts of HSV-1 positive patient samples.10 detection result of specimen show that amplification curve has obvious Exponential growth stage, all can be judged to be the positive.
Embodiment
The following example is intended to illustrate instead of limit the present invention.
Embodiment 1: the detection method of herpes simplex virus I-type test kit
(1) collection of specimens, transport and preserve
Collection of specimens:
1. depart from cell:
1.1 bleb ulcer spot scraping blades: with scraping blade scraping ulcer affected area cast-off cells, insert sterile glass tube, airtight censorship.
1.2 male genetic urinary tract secretory product cotton swabs: stretch into approximately 2～4 centimetres, urethra with tiny cotton swab, twist swab and gather secretory product, cotton swab is inserted to sterile glass tube, airtight censorship (gather secretory product and prohibit urine for first 2 hours).
1.3 female genital tract secretory product cotton swabs: wash away uterine neck ectocrine with stroke-physiological saline solution cotton balls, then insert in uterine neck with sterile cotton swab, stopped for 5 seconds after turn cotton swab gather cervical secretions, cotton swab is inserted to sterile glass tube, airtight censorship.
1.4 female urethra secretory product cotton swabs: with the clean urethral orifice of stroke-physiological saline solution cotton balls, then with approximately 2 centimetres, sterile cotton swab insertion urethra, twist swab and gather secretory product, cotton swab is inserted to sterile glass tube, airtight censorship.
2. cerebrospinal fluid: cerebrospinal fluid 0.5-1ml is extracted in standard method routinely, adds aseptic centrifuge tube, airtight censorship.
Sample is preserved and is transported: the sample gathering can be immediately for testing or being stored in for a long time-70 DEG C, and-20 DEG C of preservation perives are 6 months, and sample should be avoided multigelation.Sample transports and adopts 0 DEG C of curling stone.
(2) nucleic acid extraction
Sample to be tested and negative quality control product, HSV-1 strong positive quality control product, the critical positive quality control product of HSV-1 are carried out to synchronous processing.
A). in the aseptic centrifuge tube of 1.5ml, add the Proteinase K of 50 μ l;
B). these 200 μ l of sampling add in centrifuge tube;
C). add the cracking working fluid (containing the employing virus cracking liquid of Carrier RNA) of 200 μ l again, cover tightly pipe lid, vortex oscillation 15 seconds to be fully to mix, high speed centrifugation 10 seconds (producing bubble while preventing incubation), 72 DEG C 10 minutes.Elutriant can be placed in to 72 DEG C of preheatings simultaneously;
D). add again 250 μ l dehydrated alcohols, cover tightly pipe lid, vibrate 15 seconds;
E). mixed solution is all drawn to centrifugal column, and under room temperature 12, centrifugal 1 minute of 000rpm, is filled to new collection tube by centrifugal column;
F). the inhibition of 500 μ l is removed to liquid and add centrifugal column, under room temperature 12, centrifugal 1 minute of 000rpm, is filled to new collection tube by centrifugal column;
G). the deionization liquid of 500 μ l is added to centrifugal column, and under room temperature 12, centrifugal 1 minute of 000rpm, is filled to new collection tube by centrifugal column;
H). again the deionization liquid of 500 μ l is added to centrifugal column, under room temperature 12, centrifugal 1 minute of 000rpm, is filled to new collection tube by centrifugal column;
I). by centrifugal column-collection tube, under room temperature 14, centrifugal 3 minutes of 000rpm is to remove remaining ethanol;
J). centrifugal column is taken out, be positioned over new 1.5ml centrifuge tube.Open centrifugal column lid, place 2 minutes (use dry-type thermostat, can not use water-bath) for 72 DEG C;
K). directly over the film of centrifugal column, carefully add the elutriant 50 μ l of 72 DEG C of preheatings, cover tightly centrifugal column jecket lid, room temperature left standstill after 1 minute, centrifugal 1 minute of 14,000rpm.In centrifuge tube, be viral nucleic acid solution, suggestion is used immediately, preserves as needed, and is placed in-20 DEG C.
(3) PCR detects
A) preparation system
By 27 μ l HSV-1 reaction solution A, 3 μ l HSV-1 reaction solution B, fully mix, and then packing 30 μ l are to each PCR reaction tubes.
B) application of sample
In above-mentioned HSV-1 reaction tubes, add respectively the sample to be tested nucleic acid after extraction, negative quality control product, strong positive quality control product nucleic acid and critical positive quality control product nucleic acid 20 μ l and directly add the positive quantitatively reference material 20 μ l of centrifugal HSV-1 for subsequent use.Cover tightly pipe lid, 8,000rpm is transferred to pcr amplification instrument after the centrifugal several seconds.
C) amplification program setting
The parameter of ABI7300/7500 fluorescent PCR amplification instrument arranges as follows:
Reporter Dye1：FAM
Quencher Dye：none
Reporter Dye2：VIC
Quencher Dye：none
Passive Reference：none
The parameter of LightCycler480 fluorescent PCR amplification instrument arranges as follows:
In " Customizes " module, select FAM (483-533) and the combination of VIC (523-568) filter disc,
The unification of amplification temperature parameter arranges as follows:
50 DEG C 15 minutes;
95 DEG C 15 minutes;
40 circulations (94 DEG C 15 seconds → 55 DEG C 45 seconds), fluoroscopic examination is chosen in 55 DEG C of 45 seconds these links; Preserve file, operation.
(4) interpretation of result
Reaction finishes rear automatic saving result, according to Start value, End value and the Threshold value of image adjustment Baseline after analyzing, (user can adjust voluntarily according to practical situation, Start value can be 3～15, End value can be located at 5～20, the Value value of Threshold is set at Log collection of illustrative plates window, make baseline be positioned at amplification curve exponential phase, adjust the amplification curve of negative quality control product straight or lower than threshold line), click Analysis and automatically obtain analytical results, watch result at Report interface, record unknown sample numerical value (C).
(5) result is judged
If FAM sense channel is without increased logarithmic phase, and there is increased logarithmic phase at VIC sense channel, sentence sample and be
herpes simplex disease poisoni
typenegative.
If FAM, VIC sense channel all has increased logarithmic phase and FAM sense channel Ct value≤40; Or FAM sense channel has increased logarithmic phase and Ct value≤40, VIC sense channel is without increased logarithmic phase, sentences sample and is
herpes simplex virus I-typepositive.
Embodiment 2: the use of quality control product in herpes simplex virus I-type test kit
In herpes simplex virus I-type test kit, quality control product comprises HSV-1 strong positive quality control product, the critical positive quality control product of HSV-1, and negative quality control product, for clinical trial quality control, working method is with sample to be checked.
Negative quality control product: FAM detection path amplification curve is without increased logarithmic phase, and VIC detection path amplification curve is increased logarithmic phase;
Strong boundary positive quality control product: FAM detection path amplification curve has obvious increased logarithmic phase, and FAM sense channel amplification curve Ct value≤33;
Critical positive quality control product: FAM sense channel amplification curve has obvious increased logarithmic phase, and FAM sense channel amplification curve Ct value≤37;
Above requirement must meet in same once experiment simultaneously, otherwise it is invalid that this experiment is regarded as, need re-start.
Detected result (seeing accompanying drawing 1): the amplification curve of negative quality control product is not S-type, the amplification curve of positive quality control product is obvious S type curve, and positive and negative quality control product all meets the Quality Control requirement of test kit, and therefore the detected result of sample to be checked is effective.
Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, can also make some simple deduction or replace, all should be considered as belonging to protection scope of the present invention.
Claims (3)
1. the test kit of a fluorescent PCR detection herpes simplex virus I-type, test kit comprises: (1) nucleic acid extracting reagent, HSV-1 reaction solution A, HSV-1 reaction solution B, HSV-1 inner mark solution, negative quality control product, HSV-1 strong positive quality control product, the critical positive quality control product of HSV-1, (2) separate and concentrate and pack the bottle of these reagent or the packing box of pipe, wherein PCR reaction system comprises HSV-1 reaction solution A and HSV-1 reaction solution B, it is characterized in that in HSV-1 reaction solution A for the forward of target polynucleotide amplification and reverse primer respectively:
5’-TTATCCCATTCCTTTTGGTTCTT-3’(SEQ ID NO：1)
5’-GGGTCATGTTGGGGGCTT-3’(SEQ ID NO：2)
Oligonucleotide probe for target polynucleotide amplification is 5 '-X-TGGGGTTGGGTGGTGGAGGAGA-Y-3 ' (SEQ IDNO:3), and X wherein represents fluorescent emission group FAM, and Y is BHQ1; Oligonucleotide probe for interior mark polynucleotide amplification is 5 '-X-CCTGCTCTGACTCTGACCATCCCTG-Y-3 ' (SEQ ID NO:4), and X wherein represents fluorescent emission group HEX, and Y represents BHQ1;
Described HSV-1 reaction solution B is made up of warm start Taq enzyme, dNTPs, enzyme diluent;
Described HSV-1 inner mark solution, contain interior target area choose consistent with HSV-1 target area, and at the interior mark plasmid of one section of polynucleotide of probe location synthetic;
Described negative quality control product is physiological saline, and the critical positive quality control product of HSV-1 strong positive quality control product and HSV-1 all contains HSV-1 virus strain.
2. according to the test kit of claim 1, HSV-1 reaction solution A also comprises PCR reaction buffer, and wherein PCR reaction buffer comprises 10mmol/L Tris-HCl, 50mmol/L KCl, 3.0mmol/L MgCl
2.
3. according to the test kit of claim 1, be further characterized in that the optimal reaction temperature of pcr amplification and time is: 95 DEG C of 15min, 1 circulation; Last 94 DEG C of 15s, 55 DEG C of 45s, 40 circulations.
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| CN101824409A (en) * | 2003-04-25 | 2010-09-08 | 贝克顿·迪金森公司 | Detect 1 type and herpes simplex types 2 virus by nucleic acid amplification |
| CN102071261A (en) * | 2009-11-24 | 2011-05-25 | 上海复星医学科技发展有限公司 | Method and kit for detecting nucleic acid with herpes simplex virus |
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| CN101824409A (en) * | 2003-04-25 | 2010-09-08 | 贝克顿·迪金森公司 | Detect 1 type and herpes simplex types 2 virus by nucleic acid amplification |
| CN102071261A (en) * | 2009-11-24 | 2011-05-25 | 上海复星医学科技发展有限公司 | Method and kit for detecting nucleic acid with herpes simplex virus |
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Address after: 510665 No. 19 incense Hill Road, hi tech Industrial Development Zone, Guangdong, Guangzhou Patentee after: Guangzhou Da'an gene Co.,Ltd. Patentee after: GUANGZHOU DAAN CENTER FOR CLINICAL LABORATORY Co.,Ltd. Address before: 510665 No. 19 incense Hill Road, hi tech Industrial Development Zone, Guangdong, Guangzhou Patentee before: DA AN GENE CO., LTD. OF SUN YAT-SEN University Patentee before: GUANGZHOU DAAN CENTER FOR CLINICAL LABORATORY Co.,Ltd. |
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