CN103866049B - A kind of LAMP primer group for the I type herpes simplex virus that increases and test kit and application - Google Patents
A kind of LAMP primer group for the I type herpes simplex virus that increases and test kit and application Download PDFInfo
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Abstract
The present invention relates to biological technical field, particularly relate to a kind of LAMP primer group for the I type herpes simplex virus that increases and test kit and application.Does this primer sets comprise as SEQ? ID? 6 primers of nucleotide sequence shown in NO:1 ~ 6.Present invention also offers the test kit for detecting HSV-I virus, and non-diagnostic object detects the method for HSV-I virus.Prove through test, primer provided by the invention carries out HSV-I Viral diagnosis, and accuracy can reach 100%.And in the process utilizing common clinical poison to detect in contrast, non-false positive phenomenon produces.Further, primer provided by the invention is in HSV-I Viral diagnosis, and sensitivity can reach 10
1copies/ μ L.Be applicable to outpatient service and basic unit doctor to the quick diagnosis of the diseases such as HSV-I viral keratitis, HSV-I viral conjunctivitis, HSV-I viral retinitis.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of LAMP primer group for the I type herpes simplex virus that increases and test kit and application.
Background technology
Hsv (herpessimplexvirus, HSV) belongs to herpetoviridae α Chordopoxvirinae, and natural unique host behaves, and is divided into I type and II type.Wherein, I herpes simplex virus type (HSV-I) often causes the diseases such as herpes simplex keratitis (herpessimplexkeratitis, HSK), HSV-I viral conjunctivitis, HSV-I viral retinitis.Wherein, HSK is the primary viral keratitis of sickness rate, often can cause keratohelcosis, perforation of cornea, and blinding.Adult normal HSV seroprevalence is 90%, i.e. have HSV to hide in the gasserian ganglion of most people, when body immunity declines, the HSV hidden will cause the HSV keratitis of recurrent.
At present, conventional HSV-I detection method has: virus culture method, ELISA method, PCR method, fluorescence quantitative PCR method etc.Virus culture method requires to possess strict laboratory environment and facility, and the specialized virus culture laboratory with authorized by state could be carried out, and cost is high; And this method cycle is long, usually once cultivates and need 1-3 days, time and effort consuming, easily affect the best moment of diagnoses and treatment adversely.Although ELISA method is simple to operate, comparatively convenient, Sensitivity and Specificity is poor, easily occurs false positive.PCR method or fluorescence quantitative PCR method have good susceptibility, specificity, it is the most accurate method in current all HSV-I detection methods, but these two kinds of methods all need expensive special instrument equipment and reagent, operation steps is complicated, during laboratory fees, effort, high to personnel's technical requirements, can easily not carry out clinical.
Visible, several HSV-I detection methods conventional at present can not meet quick diagnosis or the basic medical unit use of daily outpatient service.And due to the limitation of these methods, the present situation of current clinical ophthalmology be most of patient not through the pathogenic microorganism examination accurately, the experience only relying on doctor carries out diagnoses and treatment, cause antibiotic usage more chaotic, add medical treatment cost and medical-risk.Therefore, be badly in need of high, the high specificity of a kind of susceptibility of exploitation, simple and efficient to handle, detect accurately, the method for lower-cost detection herpes simplex virus type 1.
Loop-mediated isothermal amplification technique (loop-mediatedisothermalamplification, LAMP) is the alternative PCR nucleic acid amplification method of Japanese Eiken Chemical in 2000 exploitation.The method mainly utilizes the specific region of 4 kinds of different Auele Specific Primer identification target genes, carries out amplified reaction in isothermal condition.Compared with conventional gene detection means (as PCR etc.), LAMP reaction just can complete at constant water bath box, and plant and instrument requires low, compared with normal PCR and culture method simple to operate many, do not need professional can accurately complete yet, be applicable to the application of basic medical unit.Further, LAMP can also shorten the operating time greatly, decreases sample contamination chance, is applicable to the quick diagnosis being applied to HSV-I virus.
But because the sensitivity of LAMP and specificity extremely depend on the characteristic of primer, but good LAMP primer not easily obtains, and therefore, yet there are no based on the method for LAMP technology to HSV-I viral sample direct-detection.
Summary of the invention
In view of this, technical problem to be solved by this invention is to provide a kind of LAMP primer group for the I type herpes simplex virus that increases and test kit and application, primer sensitivity provided by the invention and specificity all very high, being prepared becomes test kit and can realize detecting fast and accurately HSV-I virus.
The invention provides a kind of LAMP primer group for the HSV-I virus that increases, comprise 6 primers of nucleotide sequence as shown in SEQIDNO:1 ~ 6.
LAMP primer group provided by the invention is for the special conservative region (target gene) of HSV-I virus, be made up of 6 primers, comprise the inner primer of sequence as shown in SEQIDNO:1 ~ 2 (FIP/BIP), the outer primer of sequence as shown in SEQIDNO:3 ~ 4 (F3/B3) and the sequence ring primer (LPF/LPB) as shown in SEQIDNO:5 ~ 6.Wherein, FIP/BIP is respectively upstream and downstream internal primer, is made up of F2 district and F1C region, the F2c regional complementarity that F2 district and target gene 3 ' are held, and F1C district is identical with the Flc regional sequence that target gene 5 ' is held.F3/B3 is respectively upstream and downstream external primers, is made up of F3 district, and with the F3c regional complementarity of target gene.LPF/LPB is respectively upper and lower lantern primer, and the region that the loop-stem structure formed in amplification procedure is combined is between F2 and F1.Such design can ensure that primer has higher specificity, thus avoids occurring false positive in testing process.
Primer sets provided by the invention can be used for judging whether testing sample is subject to the pollution of HSV-1 virus.
Primer sets provided by the invention detects the application in the reagent of HSV-I virus in eye sample, skin, oral mucosa, secretory product, movement or tableware in preparation.
As preferably, eye sample is eyelid tissue, cornea tissue, tear, aqueous humor or vitreum.
Present invention also offers a kind of test kit for detecting HSV-I virus, comprising the LAMP primer group for the HSV-I virus that increases provided by the invention.
As preferably, the test kit for detecting HSV-I virus provided by the invention also comprises: LAMP reaction solution, LAMP sealing liquid and LAMP nitrite ion.
Preferably, LAMP reaction solution comprises: Bst polysaccharase, LAMP reaction buffer, ultrapure water.
Preferred, LAMP reaction buffer comprises: magnesium sulfate, trimethyl-glycine, dNTP, Tris-HCl, ultrapure water.
Preferably, LAMP sealing liquid is glycerine.
Preferably, LAMP nitrite ion comprises SYBRgreenI or fluorexon.
Present invention also offers a kind of HSV-I method for detecting virus of non-diagnostic object, comprise the following steps:
Step 1: get testing sample and mix with sterile saline, obtains sample suspension; Get primer sets as claimed in claim 1 to mix with water, obtained primer sets working fluid;
Step 2: sample thief suspension, primer sets working fluid mix with LAMP reaction solution, ultrapure water, LAMP sealing liquid, obtained amplification reaction solution;
Step 3: get amplification reaction solution, 65 DEG C of reaction 30min, whether colour developing, according to colour developing result judgement sample containing HSV-1 virus.
As preferably, colour developing adopts LAMP nitrite ion, wherein comprises SYBRgreenI or fluorexon, colour developing result present green then in testing sample containing HSV-I virus, do not present green then in testing sample containing HSV-1 virus.
Do not present green situation to comprise: colour developing result is orange, other color or colourless.
As preferably, testing sample is part tissue of eye, skin, secretory product, movement or tableware.
As preferably, in amplification reaction solution, the volume ratio of primer sets working fluid, LAMP reaction solution, sample suspension, ultrapure water and LAMP sealing liquid is 1:13.5:2:(7.5 ~ 8.5): 20.
As preferably, in primer sets working fluid,
As shown in SEQIDNO:1, the concentration of the primer of nucleotide sequence is 25 ~ 50 μm of ol/L;
As shown in SEQIDNO:2, the concentration of the primer of nucleotide sequence is 25 ~ 50 μm of ol/L;
As shown in SEQIDNO:3, the concentration of the primer of nucleotide sequence is 15 ~ 30 μm of ol/L;
As shown in SEQIDNO:4, the concentration of the primer of nucleotide sequence is 15 ~ 30 μm of ol/L;
As shown in SEQIDNO:5, the concentration of the primer of nucleotide sequence is 5 ~ 10 μm of ol/L;
As shown in SEQIDNO:6, the concentration of the primer of nucleotide sequence is 5 ~ 10 μm of ol/L.
Preferably, in primer sets working fluid,
As shown in SEQIDNO:1, the concentration of the primer of nucleotide sequence is 40 μm of ol/L;
As shown in SEQIDNO:2, the concentration of the primer of nucleotide sequence is 40 μm of ol/L;
As shown in SEQIDNO:3, the concentration of the primer of nucleotide sequence is 20 μm of ol/L;
As shown in SEQIDNO:4, the concentration of the primer of nucleotide sequence is 20 μm of ol/L;
As shown in SEQIDNO:5, the concentration of the primer of nucleotide sequence is 5 μm of ol/L;
As shown in SEQIDNO:6, the concentration of the primer of nucleotide sequence is 5 μm of ol/L.
As preferably, the volume ratio of LAMP nitrite ion and amplification reaction solution is 1:45.
The invention provides a kind of LAMP primer group for the HSV-I virus that increases, comprise 6 primers of nucleotide sequence as shown in SEQIDNO:1 ~ 6.Present invention also offers the test kit for detecting HSV-I virus, and detect the method for HSV-I virus.The present invention is based on LAMP technology, the special conservative region for HSV-I virus designs 6 primers.Therefore, primer sets susceptibility provided by the invention is high, high specificity, and the test kit be made up of it can detect the herpes simplex virus contained in testing sample fast and accurately.Further, because primer sets provided by the invention has high specificity, therefore, in LAMP amplification, directly increasing without the need to extracting the DNA of sample, further shorten the time of detection, and simplifying operation.Prove through test, primer provided by the invention carries out HSV-I Viral diagnosis, and accuracy can reach 100%.And in the process utilizing common clinical poison to detect in contrast, non-false positive phenomenon produces.Further, primer provided by the invention is in the detection of HSV-I virus, and sensitivity can reach 10
1copies/ μ L, the susceptibility of the Real-Time Fluorescent Quantitative PCR Technique the highest with current susceptibility is suitable.And due to method provided by the invention or test kit without the need to costliness instrument, also just can complete the quick and precisely detection to HSV-I virus without the need to the operation of complexity, therefore, outpatient service and basic unit doctor is applicable to the quick diagnosis of the diseases such as HSV-I viral keratitis, HSV-I viral conjunctivitis, HSV-I viral retinitis.
Accompanying drawing explanation
Fig. 1 shows the amplified production electrophoresis result of testing sample 1 ~ 3 and negative control 1 ~ 3, and wherein, swimming lane 1 ~ 3 shows the amplified production electrophoresis result of negative control 1 ~ 3, and swimming lane 4 ~ 6 shows the electrophoresis result of testing sample 1 ~ 3.
Embodiment
The invention provides a kind of LAMP primer group for the I type herpes simplex virus that increases and test kit and application.Those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
Agents useful for same of the present invention is all common commercially available product, all can buy in market.
Wherein: LAMP reaction solution, LAMP sealing liquid, LAMP nitrite ion are purchased from Guangzhou Deaou Biotechnology Co., Ltd..
Fluorescent PCR method HSV-1 kit for detecting nucleic acid is purchased from Thymopetidum Injection thing science company limited.
Other solvent and reagent are all common commercially available product.
Below in conjunction with specific embodiment, set forth the present invention further:
Embodiment 1: the accuracy of LAMP primer group provided by the invention
Utilize LAMP primer group provided by the invention to detect HSV-I virus, experiment establishes testing sample to combine negative control.LAMP primer group comprises the primer of sequence as shown in SEQIDNO:1 ~ 6.Experiment grouping is as shown in table 1:
Table 1 tests grouping and specimen in use
| Testing sample 1 | Pathological corneas place organizes | Negative control 1 | Normal cornea tissue |
| Testing sample 2 | Pathology vitreous cavity | Negative control 2 | Normal glass coelomic fluid |
| Testing sample 3 | HSV the infected's saliva | Negative control 3 | Healthy human saliva |
10 repetitions are established in each group of experiment.
LAMP experimental implementation is specially:
1, the process of clinical sample: get lesion cornea tissue and normal cornea tissue with cotton swab, fully shakes after adding 500 μ L sterile salines respectively, as far as possible by cell wash-out, fully after mixing, gets top suspension and directly can be used for LAMP reaction.Be sample suspension.
2, the preparation of working fluid:
Primer sets working fluid comprises:
As shown in SEQIDNO:1, the concentration of the primer of nucleotide sequence is 40 μm of ol/L;
As shown in SEQIDNO:2, the concentration of the primer of nucleotide sequence is 40 μm of ol/L;
As shown in SEQIDNO:3, the concentration of the primer of nucleotide sequence is 20 μm of ol/L;
As shown in SEQIDNO:4, the concentration of the primer of nucleotide sequence is 20 μm of ol/L;
As shown in SEQIDNO:5, the concentration of the primer of nucleotide sequence is 5 μm of ol/L;
As shown in SEQIDNO:6, the concentration of the primer of nucleotide sequence is 5 μm of ol/L.
Select LAMP reaction solution, LAMP sealing liquid, the LAMP nitrite ion purchased from Guangzhou Deaou Biotechnology Co., Ltd., prepare the preparation LAMP amplification system of testing sample and negative control respectively:
Following component is added respectively in little PCR pipe:
1 μ L developer is dropped in the middle of PCR pipe lid, covers tightly pipe lid.
3, the PCR pipe that testing sample and negative control LAMP amplification system are housed is put into the water-bath of 65 DEG C, react 30 minutes
4, putting upside down PCR pipe for several times, reaction solution and nitrite ion are fully mixed, observing response liquid color, if present green, be positive, and presenting orange or other colors is then feminine gender.
Experimental result shows, all presents green, and all present orange in the PCR pipe of negative control 1 ~ 3 in the PCR pipe of testing sample 1 ~ 3.Illustrate that primer provided by the invention can be differentiated in tissue accurately whether containing HSV-I virus.In order to verify the experimental result of the present embodiment further, in each PCR pipe, get 5 μ L amplified productions respectively, 2% agarose gel electrophoresis, electrophoresis 45min under 90V voltage.The amplified production electrophoresis result of testing sample 1 ~ 3 and negative control 1 ~ 3 as shown in Figure 1.As figure, after presenting the amplified production electrophoresis of green testing sample 1 ~ 3, visible scalariform amplified band, meets expection.Visible, the present embodiment is by 6 groups of experiments, and each group repeats for 10 times.60 times experimental result meets expection completely, electrophoresis result also prove colour developing result and amplification completely the same.Sufficient proof primer provided by the invention is in the process to HSV-I Viral diagnosis, and accuracy can reach 100%.
Embodiment 2: the susceptibility of LAMP primer group provided by the invention
Getting concentration is 10
4the HSV-I viral DNA of copies/ μ L, 10 times of gradient dilution samples make its titre be respectively 10
4copies/ μ L, 10
3copies/ μ L, 10
2copies/ μ L, 10
1copies/ μ L, 10
0copies/ μ L, utilizes the susceptibility of the LAMP primer group provided by the invention of the sample detection after above-mentioned dilution.
Test with the fluorescent PCR method HSV-1 kit for detecting nucleic acid purchased from Thymopetidum Injection thing science company limited as contrast.3 repetitions are established in the detection of each concentration virus.
The experimental implementation of LAMP is with reference to the embodiment of the present invention 1.
LAMP amplification system is:
Following component is added respectively in little PCR pipe:
Fluorescent PCR method detects HSV-I and operates the specification sheets provided in reference reagent box, Ct value≤36 and have good Increasing Curve of Logarithm and be judged to detect, and Ct value > 36 or amplification curve not good being judged to be do not detect.
Experimental result is as shown in table 2:
Table 2 sensitivity Detection result
Result shows, LAMP primer group provided by the invention is in the detection to HSV-I virus, and the Cmin that can accurately detect is 10
1copies/ μ L, this result is with generally acknowledged fluorescent quantitation detection method acquired results the most responsive is consistent at present.
Embodiment 3: the specificity of LAMP primer group provided by the invention
Utilize clinical common containing other viral sample respectively: adenovirus, varicella zoster virus, cytomegalovirus, HSV-2, sickle-like bacteria in streptococcus aureus, fungi and thorn ameba protozoon are as negative control, take physiological saline as blank, take HSV-I as experimental subjects, specific detection has been carried out to primer provided by the invention.LAMP experimental technique is with reference to the embodiment of the present invention 1.Establish 3 repetitions for each group.Detected result is as shown in table 3:
The specific detection result of table 3LAMP primer sets
| Repeat 1 | Repeat 2 | Repeat 3 | |
| Adenovirus | Orange | Orange | Orange |
| Varicella zoster virus | Orange | Orange | Orange |
| Cytomegalovirus | Orange | Orange | Orange |
| HSV-2 | Orange | Orange | Orange |
| Streptococcus aureus | Orange | Orange | Orange |
| Sickle-like bacteria | Orange | Orange | Orange |
| Thorn ameba protozoon | Orange | Orange | Orange |
| Physiological saline | Orange | Orange | Orange |
| HSV-I | Green | Green | Green |
Result shows, and primer sets provided by the invention can only specific amplified HSV-1, does not produce amplification, also do not produce color reaction to other pathogenic micro-organisms.Visible LAMP primer provided by the invention has good specificity, in testing process, there will not be false positive.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (9)
1. for a LAMP primer group for the HSV-I virus that increases, it is characterized in that, comprise 6 primers of nucleotide sequence as shown in SEQIDNO:1 ~ 6.
2. primer sets as claimed in claim 1 detects the application in the reagent of HSV-I virus in eye sample, skin, secretory product, oral mucosa, movement or tableware in preparation.
3. apply as claimed in claim 2, it is characterized in that, described eye sample is eyelid tissue, cornea tissue, tear, aqueous humor or vitreum.
4. for detecting a test kit for HSV-I virus, it is characterized in that, comprising LAMP primer group as claimed in claim 1.
5. test kit according to claim 4, is characterized in that, also comprises: LAMP reaction solution, LAMP sealing liquid and LAMP nitrite ion.
6. test kit according to claim 5, is characterized in that, in described LAMP reaction solution, comprising: Bst polysaccharase, LAMP reaction buffer, ultrapure water.
7. a HSV-I method for detecting virus for non-diagnostic object, is characterized in that, comprise the following steps:
Step 1: get testing sample and mix with sterile saline, obtains sample suspension; Get primer sets as claimed in claim 1 to mix with water, obtained primer sets working fluid;
Step 2: get described sample suspension, described primer sets working fluid mixes with LAMP reaction solution, ultrapure water, LAMP sealing liquid, obtained amplification reaction solution;
Step 3: get described amplification reaction solution, 65 DEG C of reaction 30min, whether colour developing, according to colour developing result judgement sample containing HSV-1 virus.
8. detection method according to claim 7, it is characterized in that, in described amplification reaction solution, the volume ratio of described primer sets working fluid, described LAMP reaction solution, described sample suspension, described ultrapure water and described LAMP sealing liquid is 1:13.5:2:(7.5 ~ 8.5): 20.
9. detection method according to claim 7, is characterized in that, in described primer sets working fluid,
As shown in SEQIDNO:1, the concentration of the primer of nucleotide sequence is 25 ~ 50 μm of ol/L;
As shown in SEQIDNO:2, the concentration of the primer of nucleotide sequence is 25 ~ 50 μm of ol/L;
As shown in SEQIDNO:3, the concentration of the primer of nucleotide sequence is 15 ~ 30 μm of ol/L;
As shown in SEQIDNO:4, the concentration of the primer of nucleotide sequence is 15 ~ 30 μm of ol/L;
As shown in SEQIDNO:5, the concentration of the primer of nucleotide sequence is 5 ~ 10 μm of ol/L;
As shown in SEQIDNO:6, the concentration of the primer of nucleotide sequence is 5 ~ 10 μm of ol/L.
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| CN101358246A (en) * | 2008-08-28 | 2009-02-04 | 中华人民共和国上海出入境检验检疫局 | LAMP kit for detecting hogcholera virus and preparation method thereof |
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| CN101358246A (en) * | 2008-08-28 | 2009-02-04 | 中华人民共和国上海出入境检验检疫局 | LAMP kit for detecting hogcholera virus and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| Hisatoshi Kaneko等.Sensitive and Rapid Detection of Herpes Simplex Virus and Varicella-Zoster Virus DNA by Loop-Mediated Isothermal Amplification.《Journal of Clinical Microbiology》.2005,第43卷(第7期),3290-3296. * |
| 潘淑静.环介导等温扩增检测单纯疱疹病毒DNA方法的建立和应用.《中国优秀硕士学位论文全文数据库医药卫生科技辑》.2009,第2009年卷(第10期),E060-107. * |
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