CN102102131A - Fluorescent PCR kit for detecting herpes simplex virus type II - Google Patents
Fluorescent PCR kit for detecting herpes simplex virus type II Download PDFInfo
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Abstract
The invention discloses a fluorescent polymerase chain reaction (PCR) kit for detecting herpes simplex virus type II, and belongs to the field of in-vitro diagnostic kit for nucleic acid. The kit comprises a positive reference, a negative reference, fluorescent polymerase chain reaction liquid, PCR primers and a specific fluorescent probe, polyethylene glycol (PEG) precipitation solution and lysis solution. The invention comprises a PCR system based on a fluorescent PCR technology, contains forward and reverse primers and the fluorescent probe for detecting the gene sequence of the herpes simplex virus type II, can detect the nucleotide sequence of the gene of the herpes simplex virus type II under proper PCR condition, can easily and quickly detect the infection of the HSV II in clinical samples and is high in specificity.
Description
Technical field
The invention belongs to external detection of nucleic acids field, relate in particular to a kind of fluorescent PCR (polymerase chain reaction) test kit that herpes simplex virus I I type (HSV-II) infects that in clinical sample, detects.
Background technology
Herpes simplex is hsv (herpes simplex virus, HSV) caused a kind of acute herpetic tetter, the people is the unique natural reservoir (of bird flu viruses) of simple scar exanthema virus, this virus is present in patient, recuperator or health carrier's blister scar liquid, saliva and the ight soil, circulation way mainly is an immediate contagion, also can be by by the tableware of saliva contamination and indirect infection.The human simple simplexvirus is divided into amphitypy, i.e. herpes simplex virus I-type (HSV-I) and herpes simplex virus I I type (HSV-II).The I type mainly causes the infection of skin, mucous membrane (oral mucosa) and organ (brain) beyond the sexual organ.The II type mainly causes the genital area mucocutaneous infections.Virus enters in the body through respiratory tract, oral cavity, sexual organ mucous membrane and damaged skin, dives to occupy in human body normal mucosa, blood, saliva and the sensory nerve ganglion cell.When Abwehrkraft des Koepers descended, during as heating, gastrointestinal dysfunction, menstruation, gestation, focal infection and mood change, the HSV that hides in the body was activated and falls ill.Hsv, the infection disease that causes is relevant with the CD4T lymphocyte number, forms ulcer pathology, herpetic whitlow refractory pathologies such as (extensively skin erosions) at lip, private parts, crissum place, and pain is obvious, also can see herpetic pneumonia, digestive tube and herpes simplex encephalitis.HSV also can cause genital herpes, and attribute spreads disease.
Hsv is mainly invaded and is originated from ectodermic tissue, as skin, mucous membrane, eye and neural system.The specific cellular immunity of human body plays an important role for limit office and the termination that HSV infects, therefore the normal person's of immunologic function primary infection often is a local infection, and immune immature newborn infant, immunologic hypofunction and height dietetic patient then can form the whole body disseminated infections.Among the patient of primary local infection also some with viremia.Humoral immunization after the primary infection can be removed big portion virus in the body, and small portion virus is often hidden in local sensory ganglion, as gasserian ganglion, superior cervical ganglion, vagus ganglion, sacral ganglia etc.In some factor, stimulate down as heating, Exposure to Sunlight, wound, menstruation, excited, operation etc., virus can be activated in neuroganglion, cause recurrence along aixs cylinder to the diffusion of the surrounding tissue of its domination, so recurrence always takes place repeatedly in same area, and the paresthesia of local skin before taking place, bleb is often arranged.This recurrence is many without viremia.
Therefore, if the pregnant woman suffers from the primary herpesvirus infection, virus just might form congenital infection by the placental infection fetus during viremia.If simplexvirus in pregnant woman's birth canal (primary infection or recurrence all can), then virus can infect the newborn infant and cause infection of newborn in birth process.No matter be congenital infection or infection of newborn, prognosis is all relatively poor, has added up 298 routine this infants if any the people, and wherein 178 examples are dead, and 57 examples have sequela, and only 66 examples are strong deposits.Therefore extremely important for the control of this illness.At present clinically the detection method of HSV is mainly contained virus culture, cytolgical examination, serum HSV-IgM type antibody test and poly synthase chain reaction (PCR) etc.Though it is to make a definite diagnosis the gold standard that HSV infects that virus culture is identified, but this method need be separated hsv and its inoculation is gone in the tissue culture medium (TCM), 24～48 hours just the pathology phenomenon can appear, length consuming time and demanding strict technology, be unfavorable for that HSV infects generaI investigation and quick diagnosis; Skin place scraping blade is that microscopically observes whether polykaryocyte and the interior eosinophilic inclusion of nuclear etc. are arranged do cytolgical examination, and this method is to carry out examination after showing clinical symptom, is unfavorable for the early diagnosis that HSV infects; The antibody test of serum HSV-IgM type easily detects false negative and false positive results, is only applicable to epidemiology survey, and is little to diagnostic value.
The fluorescent PCR technology was taken the lead in succeeding in developing by U.S. PE company in nineteen ninety-five, and it has the high sensitivity of PCR, the specificity of DNA hybridization and the advantage of spectroscopic techniques accurate quantification concurrently, and visual result can directly be monitored the variation in the PCR process.Compare with regular-PCR, but its result's Real Time Observation, product does not need detected through gel electrophoresis, and stopped pipe is operated fully, has reduced the risk of PCR product pollution effectively.
The fluorescent probe of fluorescent PCR technology forms broad variety with development, as the Taqman probe, and FRET probe etc., what present method related to is the Taqman probe technique.Its principle of work is: it can be cut active the degraded by 5 ' → 3 ' enzyme of DNA synthetic enzyme (for example Taq enzyme), 5 ' end of probe has a fluorescence report group, 3 ' end has a fluorescent quenching group, when two groups are earlier close mutually, because the transmission ofenergy effect takes place, reporter group can not send fluorescence, but carrying out along with amplified reaction, the reporter group of 5 ' end splits away off along with the hydrolysis of probe, no longer with the effect of cancellation generation transmission ofenergy, thereby can send fluorescence, be caught by signal sensor.Often the group that combines with probe 5 ' end has FAM (6-Fluoresceincarboxylic acid), TET (tetrachloro-6-Fluoresceincarboxylic acid), JOE (2,7-dimethyl-4,5-two chloro-6-Fluoresceincarboxylic acids), HEX (chlordene-6-methyl fluorescein) or VIC etc., often the fluorescent quenching group that combines with 3 ' end often is TAMRA (6-carboxyl tetramethylrhodamin) or DABCYL (4-(4 '-oxane amino-benzene azo) phenylformic acid) etc.
Summary of the invention
For overcoming that traditional method (as electrophoretic method etc.) to the shortcoming that HSV II detects, the invention provides a specific specificity height, cost is low and can making the testing product of qualitative detection.
The invention provides a kind of fluorescent PCR kit that detects herpes simplex virus I I type, comprise negative reference material, positive reference material, UNG enzyme, Taq polysaccharase, fluorescent PCR reaction solution, and DNA extraction liquid; Described fluorescent PCR reaction solution comprises PCR primer, specificity fluorescent probe.
Described positive reference material is to contain the PMD18-T vector plasmid that inserts HSV II distinguished sequence, and the distinguished sequence of described insertion is as follows:
gcgagccacatctcgcgcgcgctgttcctccccccgatcaagctcgagtgcgaaaaaacgttcaccaagctgctgctcat
cgccaagaaaaagtacatcggcgtcatctgcgggggcaagatgctcatcaagggcgtggatctggtgcgcaaaaacaact
gcgcgtttatcaaccgcacctccagggccctggtc 195bp。
Described positive reference material is 1.0 * 10 for depositing concentration
6-1.0 * 10
7The recombinant plasmid of copy/microlitre.
Described negative reference material is a PMD18-T empty carrier diluent.
Described PCR primer sequence is as follows:
The forward primer ACGTTCACCAAGCTGCTGC 19 of augmentation detection herpes simplex virus I I type;
The reverse primer CCTGGAGGTGCGGTTGAT 18 of augmentation detection herpes simplex virus I I type.
Described specificity fluorescent probe sequence is as follows:
Detect the probe TGCTCATCAAGGGCGTGGATCTGGTGC 27 of herpes simplex virus I I type.
Described specificity fluorescent probe is the Taqman probe, and what label probe 5 ' was held is a kind of fluorescence report group, can be FAM, TET, JOE, VIC, HEX, ROX, TAMRA, CY3, CY5; 3 ' end be a kind of fluorescent quenching group, can be TAMRA, DABCYL, BHQ0, BHQ1, BHQ2.
The present invention compared with prior art has the following advantages and effect:
1. quantitatively accurately, have the high sensitivity of PCR, the specificity of DNA hybridization and the advantage of spectroscopic techniques accurate quantification concurrently, visual result can be monitored the variation in the PCR process in real time.Compare with regular-PCR, but its result's Real Time Observation, product does not need detected through gel electrophoresis, and stopped pipe is operated fully, has reduced the risk of PCR product pollution effectively;
2. sensitivity and specificity height.Because take the double insurance design of specificity and primer and probe, sensitivity and specificity all improve a lot, can before occurring, clinical symptom detect the infection of virus;
3. detection speed is fast, adds that sample extraction only needs 2-3 hour altogether;
4. step is simple;
5. can carry out high-throughout pattern detection simultaneously.
Fluorescent PCR sensitivity height in a word, specificity is good, monitoring reaction process in real time, reaction times can be controlled in two and one-half-hours, and be the stopped pipe operation, need not subsequent disposal, can avoid reaction product to pollute to greatest extent, can replace conventional cell detection or regular-PCR detection the carrying out early diagnosis of HSV II.
Description of drawings:
Fig. 1 is the fluorescent PCR amplification curve of the positive reference material of HSV II: the positive reference material curve of curve, negative reference material curve;
Fig. 2 is the fluorescent PCR amplification curve of HSV II clinical sample: measured positive reference material Ct value is between 20～28, and negative reference material Ct value is Undet, and positive sample Ct value is between 20～38.
Embodiment:
Be to be understood that, unaccounted normal condition and method among the following embodiment, usually according to the conventional employing method of affiliated field experimenter: as " molecular cloning experiment guide " third edition of Sa nurse Brooker and Russell chief editor, or the step and the condition of advising according to manufacturer.
Embodiment 1: the preparation of test kit
1. primer and probe design and synthetic
Use Primer Express 3.0, at HSV II complete genome, screening is in the primer of conserved regions, the probe of design HSV II on the primer basis that chooses.Primer and probe all entrust specialized company's (giving birth to the worker) synthetic, and wherein primer is the PAGE purifying, and probe is the HPLC purifying, probe 5 ' end flag F AM fluorophor, 3 ' end mark TAMRA fluorophor.
Extension increasing sequence such as table 1:
Table 1. specific probe and primer sequence
| The sequence title | Oligonucleotide sequence (5 '-3 ') | Base length (bp) |
| HSV II probe | ?tgctcatcaagggcgtggatctggtgc | 27 |
| HSV II forward primer | ?acgttcaccaagctgctgc | 19 |
| HSV II reverse primer | ?atcaaccgcacctccagg | 18 |
2.HSV the preparation of the qualitative reference material of II plasmid positive template
HSV II plasmid is used to prepare quantitative reference material as positive template in the present embodiment.
Use synthetic amplification HSV II primer amplified HSV II gene regions target sequence in the step 1, purified PCR product is connected on the PMD18-T carrier (available from TAKARA); Be converted into then in the bacillus coli DH 5 alpha competent cell (available from TAKARA); By blue hickie screening, make up HSV II recombinant plasmid dna as positive reference material.The HSV II recombinant plasmid that makes up is identified through two-way dna sequencing.Extract plasmid, ultraviolet spectrophotometer is quantitative, and is stored in-20 ℃.
3. reference material is selected
Use no herpes simplex virus I I type to insert segmental empty carrier plasmid as negative reference material; The recombinant plasmid (1.0 * 10 that contains herpes simplex virus I I type
6-1.0 * 10
7Copy/microlitre) positive reference material.
4. the fluorescent PCR reaction solution is formed
Table 2.PCR reaction solution is formed
| Material name | Final concentration |
| PCR?Buffer | 1× |
| MgCl 2 | 2-5mM |
| dNTP | 0.1-0.5mM |
| HSV II primer | 0.1-0.50μM |
| HSV II probe | 0.1-0.50μM |
| The UNG enzyme | 0.05-1U |
| The Taq enzyme | 0.5-5U |
| H 2O | In right amount |
| The DNA masterplate | 2μL |
| Cumulative volume | 50μL |
Embodiment 2: the use of test kit
1. sample extraction
Use the HSV II nucleic acid in the DNA extraction liquid extraction testing sample
1) sample pre-treatment: wipe away clean uterine neck many secretory product of making a slip of the tongue, be close to the uterine neck oral mucous membrane with the cotton swab of physiological saline infiltration and slightly firmly rotated for 2 weeks to obtain secretory product and cast-off cells, cotton swab after the sampling is put into the fully rinsing of EP pipe that has the 1ml stroke-physiological saline solution, adherent extracting.Use the PEG precipitation, alkaline lysis method of extracting HSV II gene.
PEG precipitated liquid prescription: 30%PEG8000.
The lysate prescription: 60mmol/L TrisHCl, pH 8.0; 0.5%SDS; 200mmol/L NaCl; 1M/L NaOH.
2) operation steps: get 500 μ l secretory product and equal-volume PEG precipitated liquid concussion mixing in the centrifugal 10min of 13000rpm, abandon supernatant; Add 50 μ l lysates, 100 ℃ of insulation 20min, the centrifugal 10min of 13000rpm gets supernatant 2 μ l and does the PCR reaction.
2. pattern detection
Get the nucleic acid that extracts in the step 1 respectively, the positive reference material that obtains in the test kit, negative reference material is as dna profiling, add UNG enzyme, Taq polysaccharase and contain the specific PCR primer and the fluorescent quantitation reaction solution of specificity fluorescent probe in, form the PCR reaction system.Standard as the sample qualitative judgement.
Each main component of this system is as follows:
3. response procedures
The fluorescence detection channel of collecting the FAM fluorescent signal is set, reaction tubes is put into fluorescent PCR instrument (ABI7500) begin amplification, response procedures is as follows:
Table 3.PCR response procedures
4. the result judges
The Ct value (cycle number) of baseline scope is selected automatically for 3-15 or by software, and setting threshold makes it to surpass the maximum of random amplification curve.Fluorescent PCR instrument difference, the Ct value of gained baseline scope can be different.
5. reference material standard
(1) all kinds of control reference product product judged results such as following table:
Table 4. reference material standard testing result
| ? | Quality control product | The standard test result |
| 1 | Negative reference material | Ct value 〉=40 |
| 2 | Positive reference material | 20≤Ct value≤28 |
Fig. 1 is positive reference material and the negative reference material fluorescent PCR amplification curve diagram of HSV II.Measured positive reference material Ct value is between 20～28, and negative reference material Ct value is that Undet. (undetect does not detect) or Ct value are 40.
6. result's report:
Fig. 2 is the HSV II fluorescent PCR amplification curve of clinical sample.Measured positive reference material Ct value is between 20～28, and measured positive sample Ct value is between 20～38.
The judging criterion of sample results is as follows:
Table 4. report pattern detection result
Sequence table
＜110〉Shanghai Yue Loong Clinical Laboratory center company limited
＜120〉detect herpes simplex virus I I type fluorescent PCR kit
<160>4
<210>1
<211>195
<212>DNA
＜213〉hsv (Herpes simplex virus)
<400>1
gcgagccaca?tctcgcgcgc?gctgttcctc?cccccgatca?agctcgagtg?cgaaaaaacg 60
ttcaccaagc?tgctgctcat?cgccaagaaa?aagtacatcg?gcgtcatctg?cgggggcaag 120
atgctcatca?agggcgtgga?tctggtgcgc?aaaaacaact?gcgcgtttat?caaccgcacc 180
tccagggccc?tggtc 195
<210>2
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to nucleic acid base pairing mechanism and the design of gene chip hybridization condition, with forward primer as detection herpes simplex virus I I type fluorescent PCR kit augmentation detection HSV II.
<400>2
acgttcacca?agctgctgc 19
<210>3
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to nucleic acid base pairing mechanism and the design of gene chip hybridization condition, with reverse primer as detection herpes simplex virus I I type fluorescent PCR kit augmentation detection HSV II.
<400>3
cctggaggtg?cggttgat 18
<210>4
<211>27
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to nucleic acid base pairing mechanism and the design of gene chip hybridization condition, with probe as detection HSV II in the detection herpes simplex virus I I type fluorescent PCR kit.
<400>4
tg?ctcatcaa?gggcgtggat?ctggtgc 27
Claims (7)
1. a fluorescent PCR kit that detects herpes simplex virus I I type is characterized in that, comprises negative reference material, positive reference material, UNG enzyme, Taq polysaccharase, fluorescent PCR reaction solution, and DNA extraction liquid; Described fluorescent PCR reaction solution comprises PCR primer, specificity fluorescent probe.
2. test kit as claimed in claim 1 is characterized in that, described positive reference material is to contain the PMD18-T vector plasmid that inserts HSV II distinguished sequence, and the distinguished sequence of described insertion is as follows:
gcgagccacatctcgcgcgcgctgttcctccccccgatcaagctcgagtgcgaaaaaacgttcaccaagctgctgctcat
cgccaagaaaaagtacatcggcgtcatctgcgggggcaagatgctcatcaagggcgtggatctggtgcgcaaaaacaact
gcgcgtttatcaaccgcacctccagggccctggtc 195bp。
3. test kit as claimed in claim 1 or 2 is characterized in that: described positive reference material is 1.0 * 10 for depositing concentration
6-1.0 * 10
7The recombinant plasmid of copy/microlitre.
4. test kit as claimed in claim 1 is characterized in that: described negative reference material is a PMD18-T empty carrier diluent.
5. test kit as claimed in claim 1 is characterized in that, described PCR primer sequence is as follows:
The forward primer ACGTTCACCAAGCTGCTGC 19 of augmentation detection herpes simplex virus I I type;
The reverse primer CCTGGAGGTGCGGTTGAT 18 of augmentation detection herpes simplex virus I I type.
6. test kit as claimed in claim 1 is characterized in that, described specificity fluorescent probe sequence is as follows:
Detect the probe TGCTCATCAAGGGCGTGGATCTGGTGC 27 of herpes simplex virus I I type.
7. as claim 1 or 7 described test kits, it is characterized in that, described specificity fluorescent probe is the Taqman probe, and what label probe 5 ' was held is a kind of fluorescence report group, can be FAM, TET, JOE, VIC, HEX, ROX, TAMRA, CY3, CY5; 3 ' end be a kind of fluorescent quenching group, can be TAMRA, DABCYL, BHQ0, BHQ1, BHQ2.
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| CN102337354A (en) * | 2011-09-19 | 2012-02-01 | 泰普生物科学（中国）有限公司 | Herpesvirus II type polymerase chain reaction (PCR) fluorescent quantitative fast detection kit and method |
| CN102492699A (en) * | 2011-12-13 | 2012-06-13 | 北京英诺特生物技术有限公司 | Recombination human herpes simplex virus II protein and application thereof |
| CN103122394A (en) * | 2013-01-10 | 2013-05-29 | 湖南圣湘生物科技有限公司 | Herpes simplex virus 2 (HSV-2) detection kit |
| CN103805714A (en) * | 2014-01-08 | 2014-05-21 | 厦门安普利生物工程有限公司 | Primers, probe, kit and detection method for type II nucleic acid quantitative detection of herpes simplex virus |
| CN106916907A (en) * | 2017-05-04 | 2017-07-04 | 张家港蓝苏生物工程有限公司 | The fluorescence PCR method and kit of a kind of specific detection herpes simplex virus I, II type nucleic acid |
| CN109609699A (en) * | 2019-01-30 | 2019-04-12 | 浙江省人民医院 | A kind of kit for HSV-2 detection of nucleic acids |
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| CN110760615A (en) * | 2019-07-05 | 2020-02-07 | 北京普生诺维生物科技有限责任公司 | II type herpes simplex virus detection kit and detection method |
| CN117403010A (en) * | 2023-12-15 | 2024-01-16 | 深圳市明鉴检测专业技术有限公司 | Rapid detection method for titer of herpesvirus |
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| CN102337354B (en) * | 2011-09-19 | 2013-07-03 | 泰普生物科学（中国）有限公司 | Herpesvirus II type polymerase chain reaction (PCR) fluorescent quantitative fast detection kit and method |
| CN102337354A (en) * | 2011-09-19 | 2012-02-01 | 泰普生物科学（中国）有限公司 | Herpesvirus II type polymerase chain reaction (PCR) fluorescent quantitative fast detection kit and method |
| CN102492699A (en) * | 2011-12-13 | 2012-06-13 | 北京英诺特生物技术有限公司 | Recombination human herpes simplex virus II protein and application thereof |
| CN102492699B (en) * | 2011-12-13 | 2013-01-23 | 北京英诺特生物技术有限公司 | Recombination human herpes simplex virus II protein and application thereof |
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| CN103122394A (en) * | 2013-01-10 | 2013-05-29 | 湖南圣湘生物科技有限公司 | Herpes simplex virus 2 (HSV-2) detection kit |
| CN103805714A (en) * | 2014-01-08 | 2014-05-21 | 厦门安普利生物工程有限公司 | Primers, probe, kit and detection method for type II nucleic acid quantitative detection of herpes simplex virus |
| CN106916907A (en) * | 2017-05-04 | 2017-07-04 | 张家港蓝苏生物工程有限公司 | The fluorescence PCR method and kit of a kind of specific detection herpes simplex virus I, II type nucleic acid |
| CN109609699A (en) * | 2019-01-30 | 2019-04-12 | 浙江省人民医院 | A kind of kit for HSV-2 detection of nucleic acids |
| CN109852728A (en) * | 2019-03-13 | 2019-06-07 | 湖北朗德医疗科技有限公司 | A kind of RCA method detecting herpes simplex virus-2 (HSV-2) |
| CN110760615A (en) * | 2019-07-05 | 2020-02-07 | 北京普生诺维生物科技有限责任公司 | II type herpes simplex virus detection kit and detection method |
| CN110760615B (en) * | 2019-07-05 | 2022-04-22 | 北京普生诺维生物科技有限责任公司 | II type herpes simplex virus detection kit and detection method |
| CN117403010A (en) * | 2023-12-15 | 2024-01-16 | 深圳市明鉴检测专业技术有限公司 | Rapid detection method for titer of herpesvirus |
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Application publication date: 20110622 |