CN106834548A - A kind of common intraocular infection virus multiple real-time fluorescence PCR assay kit - Google Patents
A kind of common intraocular infection virus multiple real-time fluorescence PCR assay kit Download PDFInfo
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Abstract
The invention discloses a kind of common intraocular infection virus multiple real-time fluorescence PCR assay kit, the kit contains can be while detect the primer and probe of HSV1, HSV2, VZV, CMV and EBV virus.This kit carries out quantitative analysis to 5 kinds of common ocular infection viruses simultaneously in being reacted at one, makes up the deficiency of current detection technology.On the other hand, the detection kit may be directly applied to tissue fluid detection, so also substantially increase detection efficiency.
Description
Technical field
The present invention relates to field of biological detection, more particularly to a kind of intraocular virus multiple PCR detection techniques.
Background technology
Intraocular disease of viral infection is common ophthalmology disease, does not such as obtain timely diagnosis and treatment, and ocular tissue can be caused sternly
Damage again, or even blindness；And if can diagnose early, and impose effective treatment, majority can cure.
The intraocular virus most common pathogen of infection is mainly nerpes vinrus hominis (Human herpes virus, HHV)
Family.Nerpes vinrus hominis can be divided into 8 types, and HHV1~8 type is respectively herpes simplex virus I-form (herpes simplex
Virus, HSV- I), herpes simplex virus type II (HSV- II), varicella virus (varicella-herpes
Zoster virus, VZV), Epstein-Barr virus (EBV), human cytomegalovirus (cytomegalovirus
Retinitis, CMV), human herpes virus-6 (HHV-6), HHV-7 (HHV-7) and human herpes virus type 8
(HHV-8).That wherein most often cause intraocular virus infection is the types of HSV I and II, VZV, CMV and EBV.
Intraocular virus infection can cause the inflammation from anterior chamber of eye to posterior segment.Because intraocular virus infection can cause to regard
The huge infringement of function, therefore the antiviral therapy of early stage is a crucial step in time.And it is domestic at present for intraocular virus sense
Infectious diseases are mainly doctor and make diagnosis by patient medical history and clinical manifestation, are lacking clear and definite laboratory etiological diagnosis
Empirical antiviral therapy is used to some patientss in the case of.Even if but occur in that typical clinical manifestation,
Can not absolutely clarify a diagnosis.In fact, the intraocular disease of viral infection with typical clinical manifestations is actually rare, and have
There are plenty of such people the patient of the clinical manifestation that is not true to type, therefore clinician usually feels a delicacy about in diagnosis.If be not known
It is blindly simple to be likely to result in infection diffusion using glucocorticoid or immunodepressant, delay the state of an illness before the cause of disease.In current
State has come into the epoch of accurate medical plan, and the diagnosis and treatment of China's intraocular disease of viral infection can not be confined to again
The main suit of patient and Clinical symptom and sign, realize particularly important to the etiological diagnosis of intraocular disease of viral infection.By right
Final diagnosis, treatment effectiveness evaluation and the direction of medication usage for realizing intraocular disease of viral infection of detection of intraocular liquid virus.
The main PCR detection reagents for using commercialization of current country's intraocular viral infection illness in eye intraocular liquid detection
Box, existing kit can only detect a kind of to three kinds of herpesvirals every time.And traditional fluorescent PCR detection, all it is with disease
Malicious DNA carries out qualitative detection for sample, it is impossible to quantitative analysis is carried out to virus, and determination of the quantity of virus to therapeutic scheme has
Greatly help.Qualitative detection is carried out to DNA, then needs larger amount of tissue specimen to extract the viral DNA of q.s, otherwise can
Because DNA amount to obtain causes the result of false negative very little, and obtaining larger amount of tissue samples from eye organ has no small
Challenge.
In different part tissue of eye, the species of virus infection often has very big difference again, specific to eye fluids (anterior chamber
Water and vitreum) infection, most common Virus Type is five kinds of HSV1, HSV2, VZV, CMV and EBV.The present invention proposes a kind of new
The multiple fluorescence PCR virus detection techniques of type, it is common to 5 kinds in a reaction tube directly with sample tissue fluid as pcr template
Ocular infection virus carries out qualitative detection, and carries out virus simultaneously and carry out quantitative analysis, makes up the deficiency of current detection technology, is
A kind of fast diagnosis method for having very much an application prospect.
The content of the invention
It is an object of the invention to disclose a kind of intraocular virus multiple PCR detection kit.
The technical solution used in the present invention is：
A kind of common intraocular infection virus multiple real-time fluorescence PCR assay kit, the kit contains can be while examine
Survey the primer and probe of HSV1, HSV2, VZV, CMV and EBV virus.
Preferably, the primer of detection HSV1 is：
Forward primer：5’-CCCTCCGGCTCCCGCTCG-3’(SEQ ID NO：1)；
Reverse primer：5’-GAGGCGCCCAAGCGTCCG-3’(SEQ ID NO：2)；
Probe sequence is：5’-TTCGTCCTCGTCCTCCCCCTCCT-3’(SEQ ID NO：3).
Preferably, the primer of detection HSV2 is：
Forward primer：5’-GGTCTCGCGCGCGACCTC-3’(SEQ ID NO：4)；
Reverse primer：5’-TCGACAAGGAGGCGCCCAAG-3’(SEQ ID NO：5)；
Probe sequence is：5’-TCCCCGTCCTCGTCCCCGTC-3’(SEQ ID NO：6).
Preferably, the primer of detection VZV is：
Forward primer：5’-CGACAGACTGGGTTTTGGGTGG-3’(SEQ ID NO：7)；
Reverse primer：5’-CCGTGGGCACTGTCACAGTCTT-3’(SEQ ID NO：8)；
Probe sequence is：5’-CTGCCAACAACCCCCCATTATTACGAGT-3’(SEQ ID NO：9).
Preferably, the primer of detection CMV is：
Forward primer：5’-GGAGATGTGGTGGGGGTCAATAC-3’(SEQ ID NO：10)；
Reverse primer：5’-TCCAGGTCTTCATTGATGGGCTT-3’(SEQ ID NO：11)；
Probe sequence is：5’-TCGCTTTGAACGTAATATCGTCTGCACCTC-3’(SEQ ID NO：12).
Preferably, the primer of detection EBV is：
Forward primer：5’-CCCTCTGGACTTCCATGTCTACG-3’(SEQ ID NO：13)；
Reverse primer：5’-ACCACATACCCCTGTTTATCCGA-3’(SEQ ID NO：14)；
Probe sequence is：5’-TACACGCACGAGAAATGCGCCGT-3’(SEQ ID NO：15).
It is further preferred that the fluorophor of detection probe is at least one in FAM, VIC, TAMRA, ROX, CY5.
It is further preferred that the quenching group of detection probe is at least one in BHQ1, BHQ2, BHQ3.
The beneficial effects of the invention are as follows：Multiple PCR detection kit of the present invention can be with a reaction simultaneously to 5
Planting common ocular infection virus carries out quantitative analysis, makes up the deficiency of current detection technology.On the other hand, the detection kit can
In directly applying to tissue fluid, so also to substantially increase detection efficiency.
Brief description of the drawings
The 5 weight PCR systems of Fig. 1 the present patent application detect 5 kinds of Frozen tissue mixing samples.
The 5 weight PCR systems of Fig. 2 the present patent application detect 5 kinds of viral DNA mixing samples.
Fig. 3 control groups, prior art detects 5 kinds of Frozen tissue mixing samples.
Fig. 4 control groups, prior art detects 5 kinds of viral DNA mixing samples.
Specific embodiment
Embodiment one：
According to the various genome characteristics of HHV, the primer combination of design specific probe, invention primed probe group is shown in Table 1：
The invention primed probe group of table 1
Embodiment two
Single type Frozen tissue and standard items plasmid and 5 kinds of Frozen tissue biased samples are detected respectively with 5 weight PCR systems.
Sample type such as table 2：
Sample type added by the weight PCR system of table 25
As a result：5 weight PCR systems detect the mono- Frozen tissue of HSV1, HSV2, VZV, CMV, EBV and corresponding sun respectively
Property control plasmid, each Frozen tissue and corresponding positive plasmid all only have a kind of fluorescence channel signal there is typical case to expand in real time
Increase curve, the display mono- Frozen tissue of HSV1, HSV2, VZV, CMV, EBV and corresponding positive control plasmid all can only be unique
Detection, specificity meets the requirements.Further, 5 kinds of Frozen tissue mixing sample amplification curves, each virus amplification are detected with 5 weight PCR
Curve is normal, without template ddH2O is without obvious amplification curve.
Embodiment three：
DNA is extracted respectively to 5 kinds of single Frozen tissue samples, single Frozen tissue sample is detected simultaneously in same reaction plate
With single viral DNA sample, sensitivity of the invention and high efficiency are detected.5 kinds of single viruses are extracted using viral DNA extracts kit
Tissue DNA, its concentration is respectively HSV1 0.05ng/uL；HSV2 0.12ng/uL；VZV 0.29ng/uL, CMV 0.05ng/
UL, EBV 0.106ng/uL.Five kinds of single Frozen tissues and 5 kinds of single viral DNAs are detected respectively, as a result shows either viral group
Sample, or viral DNA sample are knitted, can there is amplification efficiency very high, judged from amplification curve Ct values, even if viral DNA is low
To 0.05ng/uL, can still obtain stabilization and expand, the display present invention either detects Virus Sample or viral DNA sample,
There is sensitivity very high.
Example IV：Viral Quantification
From the standard virus strain sample of third-party platform purchase known copy Particle density, each Virus Sample copy Particle density is shown in
Table 3.
The Virus Standard of table 3 strain copy Particle density
| Virus Standard strain | Copy Particle density (copies/uL) |
| HSV1 | |
| HSV2 | |
| VZV | |
| HCMV | |
| EBV |
To 5 kinds of virus positive control plasmid mixing, each virus particle final concentration is set to be 105Copie/uL, Ran Houjin
Row serial dilutions are 104copies/uL；103copies/uL；102copies/uL；10copies/uL.In same reaction
Plate, detects that 5 kinds of standard virus strain mixing samples mix series concentration sample with 5 kinds of positive control virus plasmids respectively using 5 weight PCR
This, calculates Average Ct values, makes standard curve, calculates each viral true copies number.Result is as shown in table 4~6.Table 4 is each disease
Malicious standard items plasmid series concentration Average Ct values；Table 5 is each virus particle standard curve linear equation；Table 6 is each Virus Standard
Strain Ct values and the starting copy number calculated according to standard curve.
45 kinds of Virus Standard quality grain series concentration Average Ct values of table
55 kinds of Virus Standard curve linear equations of table
The standard virus of table 6 strain Ct values and starting copy number
According to standard amplification curve, when plasmid concentration is 10copies/uL, still can Successful amplification, illustrate 5 kinds of viruses
Amplification efficiency can detect the as little as virus quantity of 20copies；And copy number meter is carried out with standard curve to 5 kinds of Virus Standard strains
Calculate, in error range, copy number is consistent with the viral copies Particle density of purchase, and display is the present invention can enter to sample virus number
Row is quantitative, calculates the sample virus scale of construction.
Embodiment 5：The primed probe group (invention primed probe group) that this kit is used is to tissue samples, viral DNA
Detection results and the 5 kinds of primed probes of virus of detection HSV1, HSV2, VZV, CMV, EBV (compareing primed probe to hinder) announced
Compare.
1st, sample 1：5 kinds of Frozen tissue mixtures of HSV1, HSV2, VZV, CMV, EBV
Sample 2：DNA is extracted from sample 1, that is, contains 5 kinds of mixtures of viral DNA.
2nd, sample 1 and sample 2, testing result such as table 7 and Fig. 1 are detected with control group and technical scheme respectively
Shown in~4：
Table 7 detects the Ct values for obtaining
Conclusion：
Can be obtained by Fig. 1 and Fig. 2：, in context of detection, 5 re-detections of tissue samples and DNA sample can be very for this kit
Good and specific detection.
Fig. 1 and Fig. 3 is contrasted, and Fig. 2 and Fig. 4 contrasts can be obtained：The primed probe group announced carry out 5 re-detection HSV1, HSV2,
When VZV, CMV, EBV viral DNA sample, sensitivity is below design primed probe group of the invention, detection efficiency with specificity
It is poor；And without the function of direct detection tissue samples.
SEQUENCE LISTING
<110>Guangzhou Ji Diao bio tech ltd
<120>A kind of common intraocular infection virus multiple real-time fluorescence PCR assay kit
<130>
<160> 15
<170> PatentIn version 3.5
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ctgccaacaa ccccccatta ttacgagt 28
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Claims (9)
1. a kind of common intraocular infection virus multiple real-time fluorescence PCR assay kit, it is characterised in that the kit contains
The primer and probe of HSV1, HSV2, VZV, CMV and EBV virus can simultaneously be detected.
2. detection kit according to claim 1, it is characterised in that the primer of detection HSV1 is：
Forward primer：5’- CCCTCCGGCTCCCGCTCG-3’（SEQ ID NO：1）；
Reverse primer：5’-GAGGCGCCCAAGCGTCCG-3’（SEQ ID NO：2）；
Probe sequence is：5’-TTCGTCCTCGTCCTCCCCCTCCT-3’（SEQ ID NO：3）.
3. detection kit according to claim 1, it is characterised in that the primer of detection HSV2 is：
Forward primer：5’- GGTCTCGCGCGCGACCTC -3’（SEQ ID NO：4）；
Reverse primer：5’- TCGACAAGGAGGCGCCCAAG -3’（SEQ ID NO：5）；
Probe sequence is：5’- TCCCCGTCCTCGTCCCCGTC -3’（SEQ ID NO：6）.
4. detection kit according to claim 1, it is characterised in that the primer of detection VZV is：
Forward primer：5’- CGACAGACTGGGTTTTGGGTGG -3’（SEQ ID NO：7）；
Reverse primer：5’- CCGTGGGCACTGTCACAGTCTT -3’（SEQ ID NO：8）；
Probe sequence is：5’- CTGCCAACAACCCCCCATTATTACGAGT -3’（SEQ ID NO：9）.
5. detection kit according to claim 1, it is characterised in that the primer of detection CMV is：
Forward primer：5’- GGAGATGTGGTGGGGGTCAATAC -3’（SEQ ID NO：10）；
Reverse primer：5’- TCCAGGTCTTCATTGATGGGCTT -3’（SEQ ID NO：11）；
Probe sequence is：5’- TCGCTTTGAACGTAATATCGTCTGCACCTC -3’（SEQ ID NO：12）.
6. detection kit according to claim 1, it is characterised in that the primer of detection EBV is：
Forward primer：5’- CCCTCTGGACTTCCATGTCTACG -3’（SEQ ID NO：13）；
Reverse primer：5’- ACCACATACCCCTGTTTATCCGA -3’（SEQ ID NO：14）；
Probe sequence is：5’- TACACGCACGAGAAATGCGCCGT -3’（SEQ ID NO：15）.
7. detection kit according to claims 1 to 6, it is characterised in that the fluorophor of the detection probe is
At least one in FAM, VIC, TAMRA, ROX, CY5.
8. detection kit according to claims 1 to 6, it is characterised in that the quencher of the detection probe is
At least one in BHQ1, BHQ2, BHQ3.
9. application of the detection kit in treatment of eye disorders diagnostic kit is prepared, wherein, the composition of detection kit is such as
Described in any one of claim 1~8.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108950064A (en) * | 2018-05-07 | 2018-12-07 | 北京大学第三医院 | Detect the kit and method of EBV infection in eye micro-biological sample |
| CN108950063A (en) * | 2018-05-07 | 2018-12-07 | 北京大学第三医院 | For detecting the kit and method that VZV infects in eye micro-biological sample |
| WO2019080916A1 (en) * | 2017-10-26 | 2019-05-02 | Zhuhai Qiwei Bio-Technology Ltd. | Methods and compositions for assessing and treating intraocular diseases and disorders |
| CN110241256A (en) * | 2019-05-27 | 2019-09-17 | 江苏达伯药业有限公司 | A kind of kit of multiple quantitative detection Epstein-Barr virus and cytomegalovirus |
| CN112080586A (en) * | 2020-08-20 | 2020-12-15 | 浙江大学 | Infectious ophthalmopathy pathogen solid-phase multiplex-tandem PCR detection kit and detection method |
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| WO2019080916A1 (en) * | 2017-10-26 | 2019-05-02 | Zhuhai Qiwei Bio-Technology Ltd. | Methods and compositions for assessing and treating intraocular diseases and disorders |
| US11746370B2 (en) | 2017-10-26 | 2023-09-05 | Smilebiotek Zhuhai Limited | Methods and compositions for assessing and treating intraocular diseases and disorders |
| CN108950064B (en) * | 2018-05-07 | 2021-09-24 | 北京大学第三医院 | Kit and method for detecting EBV infection in ocular trace biological sample |
| CN108950063A (en) * | 2018-05-07 | 2018-12-07 | 北京大学第三医院 | For detecting the kit and method that VZV infects in eye micro-biological sample |
| CN108950063B (en) * | 2018-05-07 | 2020-01-10 | 北京大学第三医院 | Kit and method for detecting VZV infection in ocular trace biological sample |
| CN108950064A (en) * | 2018-05-07 | 2018-12-07 | 北京大学第三医院 | Detect the kit and method of EBV infection in eye micro-biological sample |
| US12016312B2 (en) | 2018-11-14 | 2024-06-25 | Smilebiotek Zhuhai Limited | Animal models, screening methods, and treatment methods for intraocular diseases or disorders |
| CN110241256B (en) * | 2019-05-27 | 2023-07-07 | 江苏达伯药业有限公司 | Kit for multiplex quantitative detection of EB virus and cytomegalovirus |
| CN110241256A (en) * | 2019-05-27 | 2019-09-17 | 江苏达伯药业有限公司 | A kind of kit of multiple quantitative detection Epstein-Barr virus and cytomegalovirus |
| CN112080586B (en) * | 2020-08-20 | 2022-03-01 | 海南省百维恩生物科技有限责任公司 | Infectious ophthalmopathy pathogen solid-phase multiplex-tandem PCR detection kit and detection method |
| CN112080586A (en) * | 2020-08-20 | 2020-12-15 | 浙江大学 | Infectious ophthalmopathy pathogen solid-phase multiplex-tandem PCR detection kit and detection method |
| CN112195276A (en) * | 2020-10-19 | 2021-01-08 | 珞可为科技(武汉)有限公司 | Kit and method for simultaneously detecting herpes simplex virus, Kaposi's sarcoma-associated herpes virus, JC virus and EB virus |
| CN117701778A (en) * | 2024-02-01 | 2024-03-15 | 广东省人民医院 | External fuel type nano flare probe for rapidly detecting intraocular virus nucleic acid and application thereof |
| CN117701778B (en) * | 2024-02-01 | 2024-04-12 | 广东省人民医院 | External fuel type nano flare probe for rapidly detecting intraocular virus nucleic acid and application thereof |
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