CN112080586B - Infectious ophthalmopathy pathogen solid-phase multiplex-tandem PCR detection kit and detection method - Google Patents
Infectious ophthalmopathy pathogen solid-phase multiplex-tandem PCR detection kit and detection method Download PDFInfo
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Abstract
The invention discloses a solid phase multiplex tandem PCR detection kit and a detection method for infectious ophthalmopathy pathogens, which comprises the following steps: PCR mixed solution, immobilized primers and probes, positive control, negative control and RNase-free water; the solid-phase primers and probes are 8 herpesvirus primers and probes subjected to solid-phase treatment, and are respectively arranged in three-row PCR tubes, 2-3 herpesvirus primers and probes with different fluorescent labels are arranged in each PCR tube, so that the 8 herpesviruses can be simultaneously detected.
Description
Technical Field
The invention belongs to the technical field of virus detection, relates to a PCR detection kit, and particularly relates to a solid-phase multiplex tandem PCR detection kit and a detection method for infectious ophthalmopathy pathogens.
Background
Viruses, a large group of pathogens, are the most common cause of infectious eye diseases, and can cause viral keratitis (epithelia dermatitis, matritis, endophthalmitis, trabeculitis), viral uveitis, viral retinitis and the like. Clinically, the incidence of viral infectious eye disease is far higher than that of infectious eye disease caused by bacteria, fungi or other pathogens. Globally, the incidence rate of virus infectious eye diseases is high, the incidence rate is 150 to 150 of 10 ten-thousandths (1, 2) of viral keratitis alone, and 10 to 30 of 10 ten-thousandths of cases are newly increased every year (3). Recurrent episodes of viral keratitis can lead to severe scarring of the cornea and even perforation of the cornea; according to the WHO publication, corneal scarring caused by viral infection is the fourth leading cause of blindness.
So far, the detection of virus infectious eye diseases often lacks the diagnosis and treatment process based on etiology, and the missed diagnosis rate and the misdiagnosis rate are undoubtedly high. However, the treatment based on clinical observation and lack of accurate pathogen detection is more based on the anti-inflammatory treatment of the appearance rather than the targeted treatment based on the causal relationship, and cases entering the vicious circle due to misdiagnosis and mistreatment are common, so the diagnosis and treatment practice not only brings serious consequences to the health of patients, but also is not beneficial to scientifically summarizing the experience of medical staff and improving the cognition and diagnosis and treatment level. In the case of viral corneal endophthalmitis, only glaucoma-ciliary inflammation syndrome is clinically diagnosed according to the expression, and only anti-inflammatory and ocular hypotensive therapies are administered with drugs rather than viral-targeted drug therapies, which finally results in irreversible corneal endothelial decompensation and the phenomenon of corneal transplantation, and this is very common in outpatient cases, and the reasons for this are that doctors have low cognition on viral infectious eye diseases, and lack of means for pathogenic detection or methods for pathogenic detection in clinical practice are not widespread in the current situation of ophthalmology and the like. In addition, even for patients who have made diagnoses of viral infectious eye diseases based on empirical observations, blind use of antiviral drug therapy cannot achieve the purpose of accurate treatment because the kind of virus infected is unclear. Therefore, the development and application of pathogenic detection and diagnostic techniques for viral infectious eye diseases has been pressing.
To date, the vast majority of viruses that cause infectious ocular diseases are the human herpesviruses, of which the following are more common: herpes simplex virus type 1 (HSV1), herpes zoster virus (VZV), epstein-barr virus (EBV) and Cytomegalovirus (CMV), less commonly: herpes simplex virus type 2 (HSV2) and Human Herpes Virus (HHV)6,7, 8. HSV1 is the most important etiological agent of viral corneal dermatitis and stroma inflammation, and causes typical clinical manifestations of dendritic or map-like corneal epithelial defects, discoid corneal stroma edema and the like. VZV also causes corneal dermatitis and stromal inflammation, manifested as pseudodendritic epithelial defects, as well as coin-like or amorphous corneal stroma edema and cloudiness. HSV1, VZV, CMV all cause endophthalmitis [4,5], and typical symptoms are comma-like, coin-like, grid-like and linear KPs. VZV, EBV, CMV, HSV1, HSV2 and HHV-6 can cause uveitis, including anterior uveitis or iridocyclitis, as well as posterior uveitis, acute retinal necrosis or panuveitis. Viral DNA was detected in 65 specimens from 100 anterior chamber water and vitreous fluid of uveitis (including acute retinal necrosis) reported by Sugita [6], with 21 cases (21%) for VZV, 7 cases (7%) for HSV1, 6 cases (6%) for EBV and CMV, and 3 (3%) and 1 (1%) for HSV2 and HHV-6, respectively. To date, HHV-7 and HHV-8 have only been reported in a few cases [7,8.9 ]. Thus, viral keratitis and uveitis, two main components of viral infectious eye disease, can be caused by infection with different viral pathogens. Different viruses have different sensitivity and effectiveness on different antiviral drugs, such as HSV1, HSV2 and VZV, which are sensitive to acyclovir and valacyclovir, and CMV is sensitive to ganciclovir, and if pathogen detection can be carried out, the virus can not only be diagnosed clearly, but also provide basis for selecting specific and targeted antiviral drugs.
Traditional virus detection methods include live virus culture, immunohistochemical or immunofluorescence detection of viral antigens, and detection of serum virus-specific antibodies, among others. The virus culture needs a special virus culture laboratory, and has the advantages of high cost, time-consuming process and low sensitivity. The immunological antigen detecting method has low specificity and high false positive/false negative. The actual reference value of serological tests is low and is only used for speculation on the history of an infection. The virus target DNA detected by the PCR method only needs to obtain a small amount of samples, and can be used for diagnosing etiology through amplifying and detecting the target DNA, and the sensitivity and the specificity are high. PCR technology for the pathogenic diagnosis of viral infectious eye diseases began in the early nineties, and HSV and VZV DNA was detected from specimens obtained from the cornea, aqueous humor, tears and vitreous humor [10, 11, 12 ]. From the beginning of this century, PCR technology was developed rapidly, gradually from the original common PCR to real-time quantitative PCR and multiplex PCR [6,7,8 ]. Ordinary PCR can only carry out quantitative and qualitative detection on the end product of the amplification reaction. The real-time quantitative PCR can quantify the initial template, but has the defect that only one virus can be detected at one time, and if the quasi-8 viruses are screened and detected simultaneously, the technology has higher cost, time consumption and labor consumption; in addition, the amount of specimens collected from the eye is small, and the screening and detection of various viruses cannot be provided. Multiple PCR can detect a plurality of viruses in the same reaction tube simultaneously, for more than 5 viruses, because of a plurality of pairs of primers and primers, the cross reaction between the primers and the probes generates dimers, which affects the specificity and the amplification efficiency, in addition, the samples of the primers and the probes are loaded, which is tedious, time-consuming and not beneficial to popularization. Therefore, it is urgently needed to develop a technology for simultaneously detecting 8 viruses in eyes with a small sample amount, namely, a multiplex PCR technology, which has the advantages of high amplification efficiency, simple operation and low cost.
Reference to the literature
1.Liesegang TJ,Melton III LJ,Daly PJ,Ilstrup DM.Epidemiology of ocular herpes simplex. Incidence in Rochester,Minn,1950 through 1982.Arch Ophthalmol.1989；107:1155-1159.
2.Labetoulle M,Auquier P,Conrad H,et al.Incidence of herpes simplex virus keratitis in France.Ophthalmology.2005；112:888-895.
3.Young RC,Hodge DO,Liesegang TJ,Baratz KH.Incidence,recurrence,and outcomes of herpes simplex virus eye disease in Olmsted County,Minnesota,1976-2007:the effect of oral antiviral prophylaxis.Arch Ophthalmol.2010；128:1178-1183
4.Ohashi Y,Yamamoto S,Nishida K,Okamoto S,Kinoshita S,Hayashi K,Manabe R.Demonstration of herpes simplex virus DNA in idiopathic corneal endotheliopathy.Am J Ophthalmol.1991；112:419-23.
5.Koizumi N,Inatomi T,Suzuki T,Shiraishi A,Ohashi Y,Kandori M,Miyazaki D,Inoue Y,Soma T,Nishida K,Takase H,Sugita S,Mochizuki M,Kinoshita S；Japan Corneal Endotheliitis Study Group.Clinical features and management of cytomegalovirus corneal endotheliitis:analysis of 106 cases from the Japan corneal endotheliitis study.Br J Ophthalmol.2015；99:54-8.
6.Sugita S,Shimizu N,Watanabe K,Mizukami M,Morio T,Sugamoto Y,Mochizuki M.Use of multiplex PCR and real-time PCR to detect human herpes virus genome in ocular fluids of patients with uveitis.Br J Ophthalmol.2008；92:928-32.
7.Inoue T,Kandori M,Takamatsu F,Hori Y,Maeda N.Corneal endotheliitis with quantitative polymerase chain reaction positive for human herpesvirus.Arch Ophthalmol.2010；128:502-3.
8.Inoue T,Takamatsu F,Kubota A,Hori Y,Maeda N,Nishida K.Human herpesvirus 8in corneal endotheliitis resulting in graft failure after penetrating keratoplasty refractory to allograft rejection therapy.Arch Ophthalmol.2011；129:1629-30.
9.Nakano S,Sugita S,Tomaru Y,Hono A,Nakamuro T,Kubota T,Takase H,Mochizuki M,Takahashi M,Shimizu N.Establishment of Multiplex Solid-Phase Strip PCR Test for Detection of 24 Ocular Infectious Disease Pathogens.Invest Ophthalmol Vis Sci.2017；58:1553-1559.
10.Yamamoto S,Shimomura Y,Kinoshita S,et al.Detection of herpes simplex virus DNA in human tear film by the polymerase chain reaction.Am J Ophthalmol 1994；117:160–3.
11.Yamamoto S,Shimomura Y,Kinoshita S,et al.DiVerentiat-ing zosteriform herpes simplex from ophthalmic zoster.Arch Ophthalmol 1994；112:1515–6.
12.Nishi M,Hanashiro R,Mori S,et al.Polymerase chain reaction for the detection of the varicella-zoster genome in ocu-lar samples from patients with acute retinal necrosis.Am J Ophthalmol 1992；114:603–9.
Disclosure of Invention
For pathogenic diagnosis of virus-infected eye diseases, the existing method is based on conventional clinical observation, missed diagnosis and misdiagnosis are easy to occur, for example, viral corneal endophthalmitis is taken as an example, most clinical cases are easy to be diagnosed as glaucoma-cyclitis syndrome, and conventional clinical treatment only provides anti-inflammatory and intraocular pressure reduction treatment but not targeted antiviral treatment, so that corneal endothelial decompensation is easy to occur in the cases, and irreversible corneal edema is generated, so that blindness is caused. In addition, empirical observation is often too rough, and even if a virus infection is judged, the specific virus cannot be clearly identified, so that selection of an antiviral drug is very blind and lacks precise pertinence.
The invention aims to provide a solid-phase multiplex tandem PCR detection kit and a detection method for infectious ophthalmopathy pathogens aiming at the defects of the prior art, and the kit and the detection method have high sensitivity and high specificity; by applying the kit, tears are collected from the eyes of a patient, tissues, aqueous humor or vitreous humor of a cornea infected part are scraped for detection, and disease-associated viruses can be screened quickly and accurately to form etiological diagnosis and assist clinical targeted medication.
The technical scheme adopted by the invention is as follows:
infectious ophthalmopathy pathogen solid phase multiple gang PCR detection kit includes: PCR mixed solution, immobilized primers and probes, positive control, negative control and RNase-free water; the immobilized primers and probes are 8 herpesvirus primers and probes subjected to immobilized treatment, the primers and probes are respectively packaged in three-row PCR tubes, 2-3 herpesvirus primers and probes with different fluorescent labels are arranged in each PCR tube, the detection of 8 herpesviruses is realized simultaneously, and the corresponding target genes of the 8 herpesviruses are respectively as follows: the gene of HSV1 US1, HSV2 US1, VZV ORF32, EBV BDLF2, CMV UL84, HHV6 DNA polymerase activity, HHV7 UL42 and HHV8 ORF 36.
The 8 herpesvirus primers and probes are as follows:
HSV1 primer set and probe designed against US1 gene of HSV1 virus:
the upstream primer is shown as SEQ ID NO. 1;
the downstream primer is shown as SEQ ID NO. 2;
the HSV1 probe is shown as SEQ ID NO. 3; the 5 'end is marked with a fluorescence reporter group 6FAM, and the 3' end is marked with a fluorescence quenching group BQ 1;
HSV2 primer set and probe designed against US1 gene of HSV2 virus:
the upstream primer is shown as SEQ ID NO. 4;
the downstream primer is shown as SEQ ID NO. 5;
the HSV2 probe is shown as SEQ ID NO. 6; a 5 'end is marked with a fluorescent group CY5, and a 3' end is marked with a fluorescence quenching group BQ 3;
VZV primer pairs and probes designed against ORF32 gene of VZV virus:
the upstream primer is shown as SEQ ID NO. 7;
the downstream primer is shown as SEQ ID NO. 8;
the VZV probe is shown as SEQ ID NO. 9; the 5 'end is marked with a fluorescent group HEX, and the 3' end is marked with a fluorescence quenching group BQ 1;
EBV primer pairs and probes designed for the BDLF2 gene of EBV virus:
the upstream primer is shown as SEQ ID NO. 10;
the downstream primer is shown as SEQ ID NO. 11;
the EBV probe is shown as SEQ ID NO. 12; the 5 'end is marked with a fluorescent group 6FAM, and the 3' end is marked with a fluorescence quenching group BQ 1;
CMV primer pairs and probes designed against the UL84 gene of CMV virus:
the upstream primer is shown as SEQ ID NO. 13;
the downstream primer is shown as SEQ ID NO. 14;
the CMV probe is shown as SEQ ID NO. 15; a 5 'end is marked with a fluorescent group CY5, and a 3' end is marked with a fluorescence quenching group BQ 3;
HHV6 primer pair and probe designed aiming at DNA polymerase process factor gene of HHV6 virus:
the upstream primer is shown as SEQ ID NO. 16;
the downstream primer is shown as SEQ ID NO. 17;
the HHV6 probe is shown as SEQ ID NO. 18; a 5 'end is marked with a fluorescent group CY5, and a 3' end is marked with a fluorescence quenching group BQ 3;
HHV7 primer set and probe designed against UL42 gene of HHV7 virus:
the upstream primer is shown as SEQ ID NO. 19;
the downstream primer is shown as SEQ ID NO. 20;
the HHV7 probe is shown as SEQ ID NO. 21; the 5 'end is marked with a fluorescent group HEX, and the 3' end is marked with a fluorescence quenching group BQ 1;
HHV8 primer set and probe designed against ORF36 gene of HHV8 virus:
the upstream primer is shown as SEQ ID NO. 22;
the downstream primer is shown as SEQ ID NO. 23;
the HHV8 probe is shown as SEQ ID NO. 24; the 5 'end is marked with a fluorescent group HEX, and the 3' end is marked with a fluorescence quenching group BQ 1.
In the scheme, in the triple-row PCR tube, a CMV primer pair and a probe, a VZV primer pair and a probe, an HSV1 primer pair and a probe are arranged in a first tube, an HSV2 primer pair and a probe, an HHV8 primer pair and a probe are arranged in a second tube, and an HHV6 primer pair and a probe, an HHV7 primer pair and a probe, and an EBV primer pair and a probe are arranged in a third tube.
The solid phase treatment specifically comprises the following steps:
(1) dissolving 8 herpesvirus primers and probes with RNase-free water;
(2) subpackaging the PCR tubes in three rows, and subpackaging 2-3 pairs of different primers and corresponding probes in each tube;
(3) putting the PCR tube which is obtained in the step (2) and is filled with the liquid probe and the primer into an oven to evaporate water;
(4) sealing with film bag, wrapping with tinfoil, and storing at 4 deg.C.
Preferably, the mixing volume of the liquid probe and the primer is 1.2 μ L, the mixture is placed in an oven at 60 ℃ for 10min, and the probe and the primer are subjected to solid phase formation on the inner wall of the PCR tube.
The positive control is DNA cloned by 8 target genes corresponding to herpes viruses through a vector plasmid pUC57, and the negative control is physiological saline.
The solid phase multiplex tandem PCR detection method for infectious ophthalmopathy pathogens comprises the following steps:
step 1) extracting virus DNA in a sample to be detected to obtain a nucleic acid specimen;
step 2) carrying out multiple fluorescence PCR amplification by using the kit of any one of claims 1-6 and taking the positive control, the negative control and the extracted nucleic acid sample as a template;
and 3) analyzing the PCR product, and judging the virus result according to the amplification reaction result.
Further, a 20-microliter PCR reaction system is adopted in the step 2), and comprises 10-microliter PCR Mix, 8-microliter RNase-free water and 2-microliter template, wherein the concentration of each upstream primer and each downstream primer is 0.1-microliter, and the concentration of each probe is 0.2-microliter.
Preferably, the reaction procedure of the multiplex fluorescence PCR amplification is as follows: pre-denaturation: 30s at 95 ℃; denaturation: 95 ℃ for 5s, annealing extension: 30s at 65 ℃ for 45 cycles; and (3) cooling: 5s at 55 ℃.
The invention has the beneficial effects that:
the invention adopts solid phase multiplex row PCR technology, designs specific primers and a Tagman probe for detecting 8 herpes viruses, combines 3 row tubes based on the combination of different fluorescent groups of the Tagman probe, and can simultaneously detect the etiology of 8 viruses (HSV1, HSV2, VZV, EBV, CMV and HHV6,7 and 8) on an eye tissue specimen clinically suspected to be a virus infectious eye disease but not known to be a body virus. Particularly, 2-3 pairs of primers and probes of corresponding virus genes are loaded in each tube in advance by utilizing the solid-phase treatment, and only PCR Mix and a template are needed to be added in the actual use, and liquid primers or probes are not needed to be added into the PCR tubes, so that the method greatly reduces the complexity of the preparation of a multiple PCR system, shortens the preparation time of the PCR system, further saves the detection time of a sample, is simple and rapid to operate, and is suitable for the routine detection of clinical various eye virus infections; in addition, the invention adopts three-row PCR, each tube can simultaneously detect 2-3 herpes viruses, and compared with the traditional multiple PCR method, the invention can effectively reduce primer dimers and improve the amplification efficiency; but also greatly saves the specimen because: firstly, for eye specimens (tears, aqueous humor, eye tissues, etc.), the amount of collected specimens is very small compared with other body parts, the collected specimens are all used for extracting pathogens at one time, subsequent specimen retention is not needed, and secondly, the difficulty of collection is higher than that of other parts of the body (eye organs are small and precise, and nerve distribution is very abundant), so the specimens obtained by eyes are extremely precious. The method adopts the solid phase multiplex tandem PCR technology, only 3 reaction systems are needed, and only 6 microliter samples are needed, so that the sample amount is greatly saved for the extremely precious eye samples.
The kit is suitable for tears, small corneal scraping tissue, aqueous humor of the anterior chamber, vitreous humor extraction liquid or specimens of any infected part of the eye, and can be applied to virus detection of virus infected eye diseases such as viral keratitis (epithelia dermatitis, matritis and endophthalmitis), viral uveitis (including iridocyclitis, Fuchs's iridocyclitis, trichiasis syndrome, choroiditis and panuveitis), retinitis, retinal vasculitis, retinal necrosis, viral dermatitis and the like.
The technology of the invention has the advantages of high efficiency, economy, specimen saving, simple and convenient operation, easy popularization and the like which are incomparable with other prior technologies; moreover, pathogenic viruses in eight common eye infection viruses can be detected by obtaining samples such as tears, aqueous humor or vitreous humor and the like of a patient infected by the eye viruses, so that the aim of accurate and specific etiological diagnosis is fulfilled, a solid basis is provided for clinical accurate drug selection, the progress of diseases is timely and effectively controlled, the treatment effective rate and the cure rate can be improved, and the blindness rate is reduced.
Drawings
FIG. 1 is a schematic diagram showing the split charging of immobilized primers and probes in a triplet.
FIG. 2 shows the amplification curves for the 6-FAM channel positive control, HSV1(Ct value: 24.65), EBV (Ct value: 23.99).
FIG. 3 shows HEX channel amplification curves, VZV (Ct value: 25.22), HHV7(Ct value: 25.70), HHV8(Ct value: 25.10).
FIG. 4 is a Cy5 channel amplification curve, HSV2(Ct value: 25.60), CMV (Ct value: 24.34), HHV6(Ct value: 25.11).
FIG. 5 shows the results of slit lamp (a) and PCR amplification curve (b) in multiple rows on solid phase in patients with viral keratitis, which shows HSV1 positive (Ct value: 34.23).
FIG. 6 shows the results of the slit lamp (a) and the PCR amplification curve (b) in the solid-phase multiplex PCR amplification of patients with viral keratitis, which indicates CMV positivity (Ct value: 26.92).
FIG. 7 shows the results of eye slit lamp analysis (a) and solid phase multiplex PCR amplification curve (b) of a patient with trichiasis, which shows VZV positivity (Ct value: 31.12).
FIG. 8 shows the results of the solid-phase multiplex PCR amplification curve (b) and the results of the slit lamp (a) for the eye of a patient with atypical viral keratitis, which show that HHV7 is positive (Ct value: 36.18).
Detailed Description
The technical solution of the present invention is further described below with reference to specific examples.
Example 1. design combination of primers and probes for simultaneously detecting 8 human herpesviruses
The primer and probe combination designed by the invention is based on the fluorescent quantitative PCR technology, utilizes the combination of several different fluorescent groups and combines the detection capability of an instrument on the fluorescence of different channels to realize the real-time quantitative detection of a plurality of targets.
2-3 pairs of different amplification primers and corresponding Tagman probes (6FAM, HEX, CY5) with different wavelengths are placed in the same PCR tube, so that 2-3 virus targets can be detected simultaneously. Aiming at 8 herpesviruses, 3 PCR tubes are adopted, and 2-3 pairs of different primers and corresponding probes are adopted in each tube, so that the 8 herpesviruses can be screened and detected simultaneously.
The design of the primers and the probes is based on a genome database of 8 herpes viruses, and conservative and specific sequences are selected by adopting software such as MegAlign, Primer and the like. Since human genomic DNA is inevitably extracted during purification of herpesvirus DNA, primers are designed to have at least 6 base mismatches with human genomic sequence. Designing a primer pair and a probe for each herpesvirus, and finally optimizing and selecting the design combination of the primers and the probes of 8 herpesviruses by pairing dimer formation between the primers and the primers or between the primers and the probes, mutual compatibility of each combination of the primers, amplification efficiency of the primers, specificity of the primers and the invention (Table 1).
TABLE 1.8 herpesvirus primer and Probe sequences
Example 2 primer and Probe immobilization technique
The probes marked by the 8 herpes viruses are respectively as follows: 6FAM fluorescent probe (HSV1, EBV); cy5 fluorescent probe (HSV2, CMV, HHV 6); HEX fluorescent probes (VZV, HHV7, HHV 8).
The primer and the probe are immobilized on the inner wall of the PCR tube by a drying method. The 8 herpesvirus primers and corresponding probes are subpackaged into triple PCR tubes, and the herpesviruses corresponding to the primers and the probes in each tube are as follows: tubes 1 are HSV1, VZV and CMV; tube 2 is HSV2 and HHV 8; tube 3 is EBV, HHV6 and HHV7, as in FIG. 1;
the solid phase treatment comprises the following specific implementation steps:
(1) dissolving the primers and the probes by RNase-free water, and subpackaging the upstream primers and the downstream primers and the probes into corresponding PCR tubes according to the concentration required by the PCR reaction as shown in figure 1; experiments were performed using two volumes (1.2. mu.L, 2.4. mu.L).
(2) After centrifuging the PCR tube for 2min at 500g, putting the PCR tube into an oven, and evaporating water to dryness in 3 temperature modes (40 ℃, 60 ℃ and 95 ℃) to realize the solid phase of the primer and the probe.
(3) And after the solid phase is finished, sealing the film bag, and externally wrapping the film bag by using tinfoil for later use.
Under the conditions of certain volume and action temperature, the immobilization of the primers and the probes on the inner wall of the PCR tube can be realized at different times (see table 2). The temperature, time and effect are integrated, and the mixing volume of the 1.2 mu L primer and the probe is finally selected and acted for 10min at 60 ℃ as the solid phase condition.
TABLE 2 immobilization of primers and probes to the inner wall of the PCR tubes for various parameters
Example 3.8 herpesvirus solid phase tandem multiplex PCR detection kit
The PCR kit components are shown in Table 3.
TABLE 3.8 herpesvirus solid-phase tandem multiplex PCR assay kit Components
The reaction system of the kit is shown in Table 4.
TABLE 4 solid phase multiplex PCR reaction System
| 2×PCR Mix | 10μL |
| Immobilized primer probe | Solid state |
| Tear nucleic acid specimen/Positive control/negative control | 2μL |
| RNase-free water | 8μL |
| In all | 20μL |
Embodiment example 4.8 operation method and result judgment of herpesvirus solid phase tandem multiplex PCR detection kit
1. Viral nucleic acid extraction
Collecting eye specimen of patient to be diagnosed with viral ophthalmopathy, and extracting virus DNA with virus micro-extraction kit.
(1) The specimen was centrifuged at 10000 Xg for 30s, and 200. mu.L of PBS was added;
(2) adding 25 mu L of proteinase K;
(3) adding 200 mu L of lysis solution containing 6 mu g of Carrier RNA, and carrying out vortex oscillation for 15 s;
(4) placing in 56 deg.C water bath for 15min, centrifuging at 10000 Xg for 10s after water bath;
(5) adding 250 mu L of absolute ethyl alcohol, shaking for 15s, and standing for 5mm at room temperature;
(6) taking 650 mu L to a filter column, and centrifuging for 1min at 6800 Xg;
(7) adding 500 μ L of eluent, and centrifuging at 6800 × g for 1 min;
(8) repeating the step 7;
(9) centrifuging at 20000 Xg for 1min, removing residual eluate, and standing at room temperature for 1 min;
(10) add 40. mu.L RNase-free water and centrifuge at 20000 Xg for 1 min.
PCR reaction System configuration
The following experiment was carried out using the kit of example 3, where PCR Mix, specimen, positive control, and negative control were dissolved at 4 ℃ and then mixed by rapid shaking, centrifuged at 1000g for 5S, and a PCR reaction system was prepared. The reaction system configuration is shown in Table 4. The positive control was 104copy/. mu.L of the cloned DNA of the pUC57 plasmid for 2-3 herpes virus targets; the negative control was physiological saline. The concentration of the upstream primer and the downstream primer in each primer pair is 0.1. mu.M, and the concentration of each probe is 0.2. mu.M. Primer and probe sequences are shown in Table 1.
3. Amplification reaction on machine and result analysis
In this example, PCR amplification was performed using the Roche LightCycler 480II system, and the amplification procedure is shown in Table 5. The results were analyzed using a Roche LightCycler 480software 1.5.
TABLE 5 solid phase tandem multiplex PCR amplification System
4. Determination of results
(1) Baseline and threshold settings: the baseline is typically 3-15 cycles of fluorescence signal, and the threshold is set to 10 times the standard deviation of the baseline fluorescence signal.
(2) Ct value setting: number of cycles that each tube experienced when the amplified product reached the fluorescence set threshold.
(3) Positive control and negative control determination: 105copy/. mu.L positive control Ct value: HSV1(24.65), HSV2(25.60), VZV (25.22), EBV (23.99), CMV (24.34), HHV6(25.11), HHV7(25.70), HHV8(25.10). The Ct value of the negative control instrument shows that n/a, no amplification curve can be detected.
(4) And judging the clinical sample to be detected. The specific determinations are shown in Table 6.
TABLE 6 results determination of clinical specimens
Example 5.8 sensitivity evaluation of the herpesvirus solid-phase tandem multiplex PCR assay kit
The plasmid standard substance of 8 herpes virus detection targets is diluted according to a certain concentration gradient (10)5copy/μL,104copy/μL，103copy/μL，102copy/μL，101copy/. mu.L), the diluted template was amplified and the results were judged using the method of example column 4, and the results are shown in Table 7. The results show that: the R2 value of the standard curve of 8 herpesviruses is greater than 0.99, and the linear relation is good; the sensitivity of the kit was 101copy/μL，Therefore, the kit of the invention can still detect under the condition of low template concentration, and has high sensitivity.
TABLE 7.8 sensitivity detection and Standard Curve of the herpesvirus solid-phase tandem multiplex fluorescent PCR detection kit
Note: r2Representing the correlation coefficient; y represents a Ct value; x represents Log10 (copy number)
Example 6.8 specificity evaluation of the herpesvirus solid-phase tandem multiplex PCR assay kit
Samples of 8 herpesviruses (herpes simplex virus type 1/2, herpes zoster virus, epstein barr virus, cytomegalovirus, human herpes herpesvirus 6/7/8) were selected, amplified and the results determined using the method of example 2. The results show that the amplification curves of FIGS. 2-4 were not detected in any of the non-targeted viruses, indicating that the kit of the present invention has good specificity.
Example 7.8 stability evaluation of herpesvirus solid phase multiplex PCR assay kit
To evaluate the stability of the solid-phase multiplex fluorescent PCR detection kit for 8 herpesviruses, 10 samples were selected4copy/. mu.L template, 3 technical replicates each time, Ct values measured on day one, day three and day five to calculate the standard deviation and inter-group coefficient of variation within the assay groups. The results show that: for all targets and concentrations, the intraclass variability of Ct determination is less than or equal to 0.5%, and the interclass variation is less than or equal to 1%, which indicates that the solid-phase multiplex fluorescence PCR detection kit has good stability.
TABLE 8.8 evaluation of stability of the herpesvirus solid-phase tandem multiplex PCR assay kit
Note: SD, standard deviation value; CV% represents a coefficient of variation
Example 8.8 herpes virus solid phase multiplex PCR detection kit and comparison of amplification efficiency and sensitivity of single fluorescent PCR
To assess whether the solid-phase tandem multiplex technique has an effect on the amplification efficiency and sensitivity of PCR, we compared the amplification efficiency and sensitivity of solid-phase multiplex PCR with that of single-duplex fluorescent PCR. The results show that the amplification efficiency of the solid phase multiplex tandem fluorescence PCR of 8 viruses is between 80% and 120%, the sensitivity is consistent with that of the single fluorescence PCR, and the solid phase multiplex technology has no influence on the amplification efficiency and the sensitivity of the PCR.
TABLE 9 comparison of amplification efficiency and sensitivity of solid-phase multiplex PCR detection kit and single PCR
Example 9.8 herpesvirus solid phase multiplex PCR detection kit for detecting clinical specimen
The following specific procedures were performed on specimens from patients with viral keratitis, viral endophthalmitis, trichiasis syndrome, and atypical viral keratitis.
The specimen collected from viral keratitis is tear fluid. Placing 10 × 5mm sterilized filter paper on the corresponding focus part with tweezers for 30s, taking out, placing into 1.5ml centrifuge tube, placing into ice box, taking back to laboratory, storing in-80 deg.C ultra-low temperature refrigerator, and treating.
The specimen collected for viral intracorneal dermatitis and trichiasis syndrome is aqueous humor. After the patient closes the eyes, the eyelid skin is disinfected by iodophor, then the conjunctival sac is washed by levofloxacin, finally the eyes of the patient are upward seen, a needle is inserted from five points by a 1ml syringe, the patient obliquely penetrates, and about 100 mu L of aqueous humor is extracted.
The 4 samples were amplified and the results were determined using the method of example 4. The results are shown in FIGS. 5-8: viral keratitis (stromal), HSV1 positive, Ct value: 34.23; viral corneal endophthalmitis CMV-positive, Ct value: 26.92; trichiasis syndrome, positive VZV, Ct value: 31.12 of the total weight of the mixture; atypical viral corneal HHV7 positive, Ct value: 36.18. after the corresponding antiviral drugs are clinically used for the detected virus, the improvement is obvious, so that the kit has clinical popularization and application values.
The above list of embodiments is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that such changes, modifications, additions and substitutions can be made without departing from the true scope of the invention.
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Claims (5)
1. Infectious ophthalmopathy pathogen solid phase multiple gang PCR detection kit, characterized by, including: PCR mixed solution, immobilized primers and probes, positive control, negative control and RNase-free water; the immobilized primers and probes are 8 herpesvirus primers and probes subjected to immobilized treatment, the primers and probes are respectively packaged in three-row PCR tubes, 2-3 herpesvirus primers and probes with different fluorescent labels are arranged in each PCR tube, the detection of 8 herpesviruses is realized simultaneously, and the corresponding target genes of the 8 herpesviruses are respectively as follows: US1 gene of HSV1, US1 gene of HSV2, ORF32 gene of VZV, BDLF2 gene of EBV, UL84 gene of CMV, DNA polymerase process factor gene of HHV6, UL42 gene of HHV7 and ORF36 gene of HHV 8;
the 8 herpesvirus primers and probes are as follows:
HSV1 primer set and probe designed against US1 gene of HSV1 virus:
an upstream primer: 5'-TCCGGGACGTTTTCTGGATG-3', as shown in SEQ ID NO. 1;
a downstream primer: 5'-TCAGACTCCGATGAGAGGGG-3', as shown in SEQ ID NO. 2;
HSV1 probe: 5'-CCAGGCGCTTTTGCGCCTTG-3', as shown in SEQ ID NO. 3; the 5 'end is marked with a fluorescence reporter group 6FAM, and the 3' end is marked with a fluorescence quenching group BQ 1;
HSV2 primer set and probe designed against US1 gene of HSV2 virus:
an upstream primer: 5'-TCGCCACTATGGCAGACATC-3', as shown in SEQ ID NO. 4;
a downstream primer: 5'-GGGCTTACCCTCAGATTCCG-3', as shown in SEQ ID NO. 5;
HSV2 probe: 5'-CAGACGCCCCGTCCTCCCCAGCTCG-3', as shown in SEQ ID NO. 6; a 5 'end is marked with a fluorescent group CY5, and a 3' end is marked with a fluorescence quenching group BQ 3;
VZV primer pairs and probes designed against ORF32 gene of VZV virus:
an upstream primer: 5'-CCGTCGTCTATCGCACATCA-3', as shown in SEQ ID NO. 7;
a downstream primer: 5'-ATTGTACCGGCGTGTGTTCT-3', as shown in SEQ ID NO. 8;
VZV probe: 5'-TCCGTCCAAACAGTGCGCTTCA-3', as shown in SEQ ID NO. 9; the 5 'end is marked with a fluorescent group HEX, and the 3' end is marked with a fluorescence quenching group BQ 1;
EBV primer pairs and probes designed for the BDLF2 gene of EBV virus:
an upstream primer: 5'-AGCCTGTAGATTTGGGCACC-3', as shown in SEQ ID NO. 10;
a downstream primer: 5'-CAACAACACGGACAGCCTTG-3', as shown in SEQ ID NO. 11;
EBV probe: 5'-TACATGGAGGGCAGCCGCCT-3', as shown in SEQ ID NO. 12; the 5 'end is marked with a fluorescent group 6FAM, and the 3' end is marked with a fluorescence quenching group BQ 1;
CMV primer pairs and probes designed against the UL84 gene of CMV virus:
an upstream primer: 5'-CGGGTCTAGCTCCAATCGTC-3', as shown in SEQ ID NO. 13;
a downstream primer: 5'-GGGCTTACCCTCAGATTCCG-3', as shown in SEQ ID NO. 14;
CMV probe: 5'-TGTGGTTTCCGAGAGGCGCG-3', as shown in SEQ ID NO. 15; a 5 'end is marked with a fluorescent group CY5, and a 3' end is marked with a fluorescence quenching group BQ 3;
HHV6 primer pair and probe designed aiming at DNA polymerase process factor gene of HHV6 virus:
an upstream primer: 5'-TCTCGCACCACTTCTTGTCC-3', as shown in SEQ ID NO. 16;
a downstream primer: 5'-TACGAGGGTTTTGGTGACGG-3', as shown in SEQ ID NO. 17;
HHV6 probe: 5'-TGCACACAGGGAACCGACGC-3', as shown in SEQ ID NO. 18; a 5 'end is marked with a fluorescent group CY5, and a 3' end is marked with a fluorescence quenching group BQ 3;
HHV7 primer set and probe designed against UL42 gene of HHV7 virus:
an upstream primer: 5'-TAGCGTCCACCTCACTCGTA-3', as shown in SEQ ID NO. 19;
a downstream primer: 5'-GGTCGTTGCTTACTTTGCGG-3', as shown in SEQ ID NO. 20;
HHV7 probe: 5'-GCAGGCAAGGAGGATCAATGAACCC-3', as shown in SEQ ID NO. 21; the 5 'end is marked with a fluorescent group HEX, and the 3' end is marked with a fluorescence quenching group BQ 1;
HHV8 primer set and probe designed against ORF36 gene of HHV8 virus:
an upstream primer: 5'-CTTGCGTCCTCTTCCAGTGT-3', as shown in SEQ ID NO. 22;
a downstream primer: 5'-AGGGCTGATAGGGTACGGTT-3', as shown in SEQ ID NO. 23;
HHV8 probe: 5'-TGCACGCGCAGGTATTGGACC-3', as shown in SEQ ID NO. 24; the 5 'end is marked with a fluorescent group HEX, and the 3' end is marked with a fluorescence quenching group BQ 1.
2. The solid phase multiplex tandem PCR assay kit for infectious ophthalmopathy pathogens according to claim 1, wherein the triple tandem PCR tubes comprise CMV primer pair and probe, VZV primer pair and probe, HSV1 primer pair and probe in the first tube, HSV2 primer pair and probe, HHV8 primer pair and probe in the second tube, and HHV6 primer pair and probe, HHV7 primer pair and probe, EBV primer pair and probe in the third tube.
3. The solid-phase multiplex tandem PCR detection kit for infectious ophthalmopathy pathogens according to claim 1, wherein the solid-phase treatment specifically comprises:
(1) dissolving 8 herpesvirus primers and probes with RNase-free water;
(2) subpackaging the PCR tubes in three rows, and subpackaging 2-3 pairs of different primers and corresponding probes in each tube;
(3) putting the PCR tube which is obtained in the step (2) and is filled with the liquid probe and the primer into an oven to evaporate water;
(4) sealing with film bag, wrapping with tinfoil, and storing at 4 deg.C.
4. The kit for detecting the pathogen of infectious eye diseases by solid-phase multiplex tandem PCR as claimed in claim 3, wherein the volume of the mixture of the liquid probe and the primers is 1.2 μ L, the mixture is placed in an oven at 60 ℃ for 10min, and the probe and the primers are immobilized on the inner wall of the PCR tube.
5. The solid-phase multiplex row PCR detection kit for the infectious ophthalmopathy pathogens according to claim 1, wherein the positive control is DNA cloning of 8 target genes corresponding to the herpesviruses by a vector plasmid pUC57, and the negative control is normal saline.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101351559A (en) * | 2005-11-09 | 2009-01-21 | 普里梅拉生物系统有限公司 | Multiplexed quantitative detection of pathogens |
| CN103045761A (en) * | 2012-12-14 | 2013-04-17 | 山东省眼科研究所 | Kit and detection method for detecting pathogenic microorganisms of infectious eye disease |
| CN106834548A (en) * | 2017-03-30 | 2017-06-13 | 广州基迪奥生物科技有限公司 | A kind of common intraocular infection virus multiple real-time fluorescence PCR assay kit |
| WO2017106616A1 (en) * | 2015-12-17 | 2017-06-22 | The Regents Of The University Of Colorado, A Body Corporate | Varicella zoster virus encoding regulatable cas9 nuclease |
-
2020
- 2020-08-20 CN CN202010842872.8A patent/CN112080586B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101351559A (en) * | 2005-11-09 | 2009-01-21 | 普里梅拉生物系统有限公司 | Multiplexed quantitative detection of pathogens |
| CN103045761A (en) * | 2012-12-14 | 2013-04-17 | 山东省眼科研究所 | Kit and detection method for detecting pathogenic microorganisms of infectious eye disease |
| WO2017106616A1 (en) * | 2015-12-17 | 2017-06-22 | The Regents Of The University Of Colorado, A Body Corporate | Varicella zoster virus encoding regulatable cas9 nuclease |
| CN106834548A (en) * | 2017-03-30 | 2017-06-13 | 广州基迪奥生物科技有限公司 | A kind of common intraocular infection virus multiple real-time fluorescence PCR assay kit |
Non-Patent Citations (2)
| Title |
|---|
| Establishment of Multiplex Solid-Phase Strip PCR Test for Detection of 24 Ocular Infectious Disease Pathogens;Nakano Satoko等;《Investigative ophthalmology & visual science》;20170301;第58卷(第3期);第1553-1559页 * |
| 病毒感染性眼病病原学诊断的研究进展;段润平等;《浙江大学学报(医学版)》;20200723;第49卷(第5期);第644-650页 * |
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