PCR技术在单纯疱疹病毒检测中的应用进展

单纯疱疹病毒是人类病毒性疾病中最常见的病毒,PCR技术以其高灵敏度、高特异性、快速、简便等优点,为单纯疱疹病毒的检测提供了新途径.现就该方法在单纯疱疹病毒检测中的应用及进展作一综述.     

Unlabeled probes for the detection and typing of herpes simplex virus

BACKGROUND: Unlabeled probe detection with a double-stranded DNA (dsDNA) binding dye is one method to detect and confirm target amplification after PCR. Unlabeled probes and amplicon melting have been used to detect small deletions and single-nucleotide polymorphisms in assays where template is in abundance. Unlabeled probes have not been applied to low-level target detection, however. METHODS: Herpes simplex virus (HSV) was chosen as a model to compare the unlabeled probe method to an in-house reference assay using dual-labeled, minor groove binding probes. A saturating dsDNA dye (LCGreen Plus) was used for real-time PCR. HSV-1, HSV-2, and an internal control were differentiated by PCR amplicon and unlabeled probe melting analysis after PCR. RESULTS: The unlabeled probe technique displayed 98% concordance with the reference assay for the detection of HSV from a variety of archived clinical samples (n = 182). HSV typing using unlabeled probes was 99% concordant (n = 104) to sequenced clinical samples and allowed for the detection of sequence polymorphisms in the amplicon and under the probe. CONCLUSIONS: Unlabeled probes and amplicon melting can be used to detect and genotype as few as 10 copies of target per reaction, restricted only by stochastic limitations. The use of unlabeled probes provides an attractive alternative to conventional fluorescence-labeled, probe-based assays for genotyping and detection of HSV and might be useful for other low-copy targets where typing is informative.     

Evaluation of the Cepheid herpes simplex virus typing real-time PCR assay using dermal and genital specimens

The Cepheid herpes simplex virus (HSV) (Cepheid, Sunnyvale, CA) typing multiplex real-time polymerase chain reaction (PCR) assay was evaluated for its ability to detect HSV in dermal and genital specimens stored in M5 media. Swab specimens (n = 114) for HSV testing were placed in M5 media and split between our laboratory and a highly experienced reference laboratory. Aliquots for testing with the Cepheid assay were processed using a simple boil-and-go procedure and then run in a SmartCycler II (Cepheid). Aliquots tested at the reference laboratory were processed using a MagNA Pure LC DNA extractor (Roche Molecular Systems, Alameda, CA) and tested by the Roche HSV real-time PCR assay. Both laboratories detected 35 positives. Of the positive specimens, the Cepheid assay typed 16 as HSV 1 and 19 as HSV 2; the reference laboratory typed 15 as HSV 1, 19 as HSV 2, and 1 as HSV indeterminate. Our results demonstrate that the Cepheid real-time PCR assay, using specimens subjected to minimal specimen processing, performed as well as the Roche real-time PCR assay, using DNA extracts, for the detection of HSV DNA in genital and dermal specimens.     

Comparison of four methods for typing low-passage herpes simplex virus isolates

Clinical isolates of herpes simplex virus (HSV) were identified as HSV type 1 or type 2 by sensitivity to (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), by differential replication in chick embryo cells versus guinea pig embryo cells, by restriction endonuclease analysis, and by a direct fluorescent antibody technique using monoclonal antibodies. More than 550 isolates were typed by two or three of the systems with complete agreements as to virus type between systems for each isolate. In appropriately equipped laboratories, any of the above typing systems can be used with complete confidence. However, the BVDU sensitivity assay, particularly when used in a continuous cell line as described, can be economically utilized in any virology laboratory.     

A novel protein tag from herpes simplex virus type 1 DNA polymerase.

An epitope (HPOL) derived from the so-called thumb region of the herpes simplex virus type 1 DNA polymerase in combination with a monoclonal antibody (MAb 1051c) was tested for protein tagging. Using a conventional expression vector, a DNA cassette encoding the HPOL epitope was fused to the C-terminus of the dihydrofolate reductase (DHFR) gene such that the recombinant DHFR contained both a N-terminal HIS-tag and a C-terminal HPOL tag. Expression of recombinant DHFR in Escherichia coli cells was compared by Western blot analysis using either mouse RGS.HIS antibody or MAb 1051c. Immunostaining revealed that both antibodies reacted specifically with DHFR, but the detection sensitivity achieved with MAb 1051c was about 15-fold greater using a standard staining protocol. An HPOL antibody column was successfully applied for affinity purification of DHFR, demonstrating the usefulness of the HPOL epitope/MAb 1051c system for protein tagging, expression monitoring and purification of HPOL-tagged recombinant proteins.     

A flexible bioluminescent-quantitative polymerase chain reaction assay for analysis of competitive PCR amplicons

Aequorin-based flash-type bioluminescent methods can detect nucleic acid molecules in the attomolar range (10(-18)) enabling improved monitoring of the polymerase chain reaction (PCR) at cycles previously considered too low for product detection. The high sensitivity of bioluminescence (BL) was used to examine the efficiency of the PCR and to assess the effect of substrate variation during the linear phase of amplification. Primer efficiency was dependent on initial template concentration, in a manner indicative of a two-component reaction. However, the rate of amplicon formation was significantly impaired at low template levels and could not be overcome by excess primer. The PCR was directly dependent upon nucleotide concentration, which was independent of template concentration. Conditions were identified for optimal linear amplification and detection using BL. Accurate quantitative analysis was performed using competitive coamplification of a specific target standard sequence containing identical target primer recognition sites and novel internal sequences. Quantitation was most accurate when target molecule was similar in concentration to the internal standard. The Bioluminescent Quantitative-PCR (BLQ-PCR) assay has the potential to eliminate processing variability. We demonstrated high quantitative potential with a broad dynamic range. Overall, the BLQ-PCR assay is flexible and a viable alternative to contemporary Q-PCR techniques.     

DNA polymerase mutations in drug-resistant herpes simplex virus mutants determine in vivo neurovirulence and drug-enzyme interactions

Mutations in the thymidine kinase and DNA polymerase genes of herpes simplex virus (HSV) might confer resistance to antiviral drugs, particularly in immunocompromised patients who suffer from chronic and/or disseminated lesions. The patterns of cross-resistance and neurovirulence in mice of several DNA polymerase mutants selected under pressure of foscarnet (PFA) and different acyclic nucleoside phosphonates (ANPs), including (S)-3-hydroxy-2-phosphonylmethoxypropyl (HPMP) derivatives of adenine (HPMPA) and cytosine (HPMPC, cidofovir) and 2-phosphonylmethoxyethyl (PME) derivatives of adenine (PMEA) and 2,6-diaminopurine (PMEDAP), were investigated. The mutants were derived from the HSV-1 strain KOS following either single or multiple steps of selection with PFA (V714M, A719V, 5724N and T821M), PMEA (S724N, L802F and R959H), PMEDAP (Q618H, S724N, S724N+D1070N), HPMPC (V573M, R700M and K960R) or HPMPA (W998L, L1007M and 11028T). These amino acid substitutions were located in different subdomains of the HSV-1 DNA polymerase, either in conserved or non-conserved regions. The sensitivity of the mutants to a new class of ANPs, the 6-(2-[phosphonomethoxy]alkoxy)pyrimidines HPMPO-DAPy and PMEO-DAPy, was investigated. Cross-resistance between the HPMP derivatives and HPMPO-DAPy, on the one hand, and between the PME derivatives and PMEO-DAPy, on the other hand, was observed. Different degrees of cross-resistance between PME derivatives, PMEO-DAPy, PFA and acyclovir were noticed. The mutants ranged from exhibiting near wild-type neurovirulence (V714M, A719V, 5724N and L1007M) to significant attenuation (Q618H, S724N+D1070N, L802F, R700M, K960R, W998L and 11028T) or higher levels of attenuation (V573M). It appears that drug-resistant mutants arising under the pressure of HPMP derivatives have the lowest levels of neurovirulence.     

Evaluation of a new herpes simplex virus typing reagent for tissue culture confirmation

Monoclonal antibody typing reagents for herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) from California Integrated Diagnostics, Inc. (Berkeley, CA) were compared to two other commercially available HSV monoclonal antibody typing reagents. Of the 105 specimens tested, of which 81 were positive for HSV, there was 100% agreement with all three typing reagents.     

Variability of the region of the herpes simplex virus type 1 genome yielding defective DNA: SmaI fragment polymorphism

A SmaI map of class I defective DNA of herpes simplex virus type 1 (HSV1) was constructed using a set of deletion hybrid phages. Four SmaI fragments on the defective DNA had a variability in length common among 15 HSV1 isolates: the 1.45 kilobase (kb) fragment located within the BamHI-Z (map coordinates 0.936-0.949) fragment, the 0.92-kb fragment neighboring on the 'a' sequence, the 0.44-kb fragment containing the intervening sequence of immediate-early mRNA-5 gene, and the 0.205-kb fragment corresponding to the 'a' sequence. The four SmaI fragments have several sets of reiterated sequences, among which the 1.45- and 0.92-kb fragments hybridized with mammalian cellular DNAs (human, monkey, rabbit, and mouse).     

Detection of herpes simplex virus by biotinylated DNA probes

Clinical specimens from 159 patients suspected with herpes simplex virus (HSV) were examined by monoclonal antibody immunofluorescence (IF) and by a commercial biotinylated DNA probe kit following cell culture isolation. Herpes simplex virus was isolated from 57 samples. All cultures were positive by IF when the cytopathic effect (CPE) was less than 1+ but only 49 (86%) yielded positive reaction with the DNA probe when CPE was at least 1+. A total of 54 clinical specimens was also examined directly by immunoperoxidase histopathology (IHP), IF, and DNA hybridization. Of these, 16 were positive by IHP, 15 by IF, and only five by DNA probe. The DNA probe kit was found to be reasonably sensitive only after cell culture isolation of HSV. Compared to the IF procedure, the DNA probe kit was found to be costly, labor intensive, and time consuming.     

Tetracycline-regulated gene expression in replication-incompetent herpes simplex virus vectors

Although herpes simplex virus (HSV) vectors appear to have great potential as gene delivery vectors both in vitro and in vivo, the expression of foreign genes in such vectors cannot be easily regulated. Of the known eukaryotic regulatory systems, the tetracycline-inducible gene expression system is perhaps the most widely used because of its induction characteristics and because of the well-known pharmacological properties of tetracycline (Tet) and analogs such as doxycycline. Here, we describe the adaptation of the Tet-inducible system for use in replication-incompetent HSV vectors. HSV vectors were constructed that contained several types of Tet-inducible promoters for foreign gene expression. These promoters contained a tetracycline response element (TRE) linked to either a minimal cytomegalovirus (CMV) immediate-early promoter, a minimal HSV ICP0 promoter, or a truncated HSV ICP0 promoter containing one copy of the HSV TAATGARAT cis-acting immediate-early regulatory element (where R represents a prime base). All three promoter constructs were regulated appropriately by doxycycline, as shown by the expression of the marker gene lacZ in cell lines engineered to express Tet transactivators. The ICP0 promoter constructs expressed the highest and most sustained levels of lacZ, but the CMV promoter construct had the highest relative level of induction, suggesting their use in different applications. To extend the utility of Tet-regulated HSV vectors, vectors were constructed that coexpressed an inducible Tet transactivator in addition to the inducible lacZ marker gene. This modification resulted in tetracycline-inducible gene expression that was not restricted to specific cell lines, and this vector was capable of inducible expression in irreversibly differentiated NT2 cells (NT-neurons) for several days. Finally, HSV vectors were constructed that expressed modified Tet transactivators, resulting in improved induction properties and indicating the flexibility of the Tet-regulated system for regulation of foreign gene expression in HSV vector-infected cells.     

Replicating DNA of herpes simplex virus type 1.

Newly synthesized herpes simplex virus type 1 DNA yielded a heterogeneous sedimentation profile in neutral sucrose gradients, with the main peak occurring at approximately 40S. Components sedimenting slower than virion DNA and a rapidly sedimenting intracellular HSV DNA were also observed. Both the low-molecular weight and the rapidly sedimenting components seemed to be precursors of virion DNA: they almost completely disappeared after a 60-min chase of a 3-min pulse of 3H-thymidine, and were converted into DNA which cosedimented with virion 32P-labeled DNA. However, sedimentation analysis in alkaline sucrose gradients showed that a 60-min period was insufficient for completing the maturation of HSV DNA. Cleavage of parental DNA molecules was observed in neutral sucrose gradients after infection with 3H-thymidine-labeled virions. No evidence for the formation of covalently closed circles during the replication process was obtained. The presence of single-stranded regions in the replicative form of HSV DNA was revealed. Some of the short-pulse (30 sec) labeled HSV DNA (26.1%) was eluted from hydroxylapatite columns with the properties of single-stranded DNA, and 22% of its trichloroacetic acid precipitability was susceptible to single-strand specific S1 nuclease treatment. Pulse-chase experiments indicated that the life-time of this single-stranded component in nascent DNA was probably not longer than 3 min. A small proportion of single-stranded regions, however, survived for longer periods. Almost all of the newly synthesized short-pulse-labeled HSV DNA exhibited an affinity for nitrocellulose filters. This affinity, which was S1 nuclease-sensitive, gradually decreased with prolongation of the time of the chase. After chasing the pulse for 1 h, the attachment of newly synthesized DNA was comparable with virion DNA.     

Evaluation of the ELVIS plate method for the detection and typing of herpes simplex virus in clinical specimens

This study was conducted to assess the reliability of a commercial enzyme-linked viral inducible system (ELVIS) (Diagnostic Hybrids, Inc., Athens, OH) for rapid detection and typing of herpes simplex virus (HSV). Results using ELVIS were compared to those of shell vial culture (SVC) and HSV detection with monoclonal antibodies and an immunoperoxidase stain plus typing with MicroTrak direct fluorescent antibodies (Trinity Biotech PLC, Wicklow, Ireland). Specimens yielding discrepant HSV results were tested by polymerase chain reaction (PCR); those with discrepant typing results were stained with Simulfluor (Chemicon, Temecula, CA). Of the 206 samples tested, 144 were negative and 54 were HSV-positive by both methods (agreement, 96.1%). Five specimens were positive by ELVIS but negative by SVC; 3 of these were positive and 2 were negative by HSV PCR. Both of the latter were the result of mechanical problems early in the study. Three specimens were positive by SVC but negative by ELVIS; all 3 were positive by HSV PCR. After resolution of discrepancies, the sensitivity and specificity for detection of HSV were 95.0% and 100% for SVC, respectively, and 95.0% and 98.6% for ELVIS. Of the 46 HSV-positive samples that were typed, 26 were called type 2 and 18 were type 1 by both methods (agreement, 95.7%). The 2 specimens with discrepant results were called HSV-2 by SVC, staining with MicroTrak, and HSV-1 with ELVIS; both of these were type 2 when stained with the Simulfluor reagent. ELVIS is a reliable alternative to SVC for rapid detection and typing of HSV.     

Stability of the cloned 'joint region' of herpes simplex virus DNA

To isolate stable recombinants containing the 'joint region', or L-S junction, of herpes simplex virus DNA, the EcoRI restriction enzyme cleavage fragments were cloned into both coliphage lambda and plasmid vectors. The authentic joint region was found in the plasmid but not in the lambda vector. The plasmid-joint region recombinant DNAs appeared stable on limited passage. Subcloning the small BamHI L-S junction fragment into plasmid pBR322 gave rise to both stable and unstable recombinant DNAs.     

Evaluation of three analyte-specific reagents for detection and typing of herpes simplex virus in cerebrospinal fluid

The performance of three analyte-specific reagents (ASRs); Cepheid herpes simplex virus (HSV) Typing Primer Probe set (CD), Eragen MultiCode-Rtx HSV-1/2 kit primer mix (ER), and Roche LightCycler HSV-1/2 Primer/Hybridization Probes (RD), was evaluated for detection and typing of herpes simplex virus (HSV-1 and HSV-2) in cerebrospinal fluid (CSF) specimens. Of 68 CSF specimens, HSV-1 was detected in 8 specimens and HSV-2 was detected in 17 specimens. ER detected all 25 HSV-positive specimens, whereas CD and RD detected 24 and 23 HSV-positive specimens, respectively. The results of HSV typing with the 3 ASRs were in complete agreement. The analytical sensitivity of all ASRs was determined to be about 10¹ copies/reaction. Our results demonstrate that the performances of all 3 ASRs are comparable and reliable for routine clinical testing in detection and typing of HSV DNA in CSF.     

Inhibition of cardiolipin synthesis following infection with herpes simplex virus

The results of this study show that the synthesis of the inner mitochondrial membrane phospholipid, cardiolipin, is markedly inhibited following infection of human embryonic lung or hamster embryo fibroblast cells with herpes simplex virus type 1 or 2. The synthesis of other phospholipids, i.e., phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and phosphatidylinositol, is relatively unaffected.