A duplex PCR assay for detection and genotyping of Herpes simplex virus in cerebrospinal fluid

Molecular and Cellular Probes (1999) 13, 309–314 Article No. mcpr.1999.0253, available online at http://www.idealibrary.com on

A duplex PCR assay for detection and genotyping of Herpes simplex virus in cerebrospinal fluid L. I. Gonza´lez-Villasen˜or∗ BBI-Biotech Research Laboratories, Gaithersburg, MD 20877, USA (Received 9 April 1999, Accepted 25 May 1999) A duplex polymerase chain reaction (PCR) assay for the detection and genotyping of Herpes simplex virus (HSV) 1 and 2 from cerebrospinal fluid (CFS) of infants was developed. The glycoprotein D (gD) gene of HSV was selected as a target for amplification. The assay is highly specific, sensitive and reproducible. Herpes simplex virus detection is performed by agarose gel electrophoresis and Southern blot using a chemiluminescent probe. The probe hybridizes to sequences common to both HSV-1 and 2. A DNA fragment of HSV gD gene was cloned and used as positive control and to determine the specificity and sensitivity of the assay. The PCR assay is user-friendly and unambiguously differentiates in one-step both herpes virus strains. The assay is useful to screen CFS specimens from infants exposed to HSV during birth and at risk of developing encephalitis.  1999 Academic Press

KEYWORDS: Herpes simplex virus, genotyping, PCR, cerebrospinal fluid, encephalitis, infants.

INTRODUCTION

Herpes simplex virus (HSV) infections in humans has been recognized as a sexually transmitted disease (STD) since the eighteenth century.1,2 The virus contains 11 surface glycoproteins and a double-stranded DNA genome of approximately 150 kbp with a high (68%) G-C content.3 Herpes viruses are classified into two types depending on the way the infections are acquired. HSV-1 or labial herpes is acquired by contact with salivary secretions and replicates in the oropharyngeal mucosa. HSV-2 or genital herpes is acquired almost exclusively genitally and replicates in the genital areas. Both agents belong to the Herpesviridae family, have a worldwide distribution and are closely related.3 Herpes simplex virus has two unique properties: its virulence or ability to cause disease, and its latency or ability to remain latent in the human host’s lifetime.3 Herpes simplex virus infections in humans occur by

infecting superficial tissues and establishing an acute infection. During this period HSV infects innervating neurons, spreads up their axons into sensory ganglia and establishes in sensory neurons.4 A large variety of infectious micro-organisms including HSV-1 and 2 can cause central nervous system (CNS) infections. One major CNS infection is encephalitis. Neonatal encephalitis due to HSV infection is a life-threatening disease and difficult to diagnose because HSV encephalitis (HSVE) can mimic other CNS disorders.5 Nearly one-third of all babies with neonatal HSV infection have encephalitis only.6 About 50% of these babies die due to untreated localized CNS disease.7 Although cell culture and serological techniques have been used to detect HSV, they are not reliable. Culture of cerebrospinal fluid (CSF) is usually negative.8 Detection of HSV-1 and 2 using Western blot,9 monoclonal antibodies,10,11 immunostaining12 and IgG and IgM antibody-antigen tests,13 have also been used to detect HSV. However, antigen-antibody assays are

∗ Address for correspondence: National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

0890–8508/99/040309+06 $30.00/0

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not useful because they develop 3–10 days after onset of neurological symptoms5 and cannot differentiate between HSV-1 and 2. New sensitive and specific tests are thus essential for proper diagnosis of encephalitis and meningitis in newborns directly from CSF samples within the first days of infection.14,15 Molecular assays such as the polymerase chain reaction (PCR) have been developed to detect HSV in a variety of specimens including CSF.16,17 Many PCR assays have targeted the genome’s short (US)18 and long (UL) component,19 the DNA polymerase gene,20,21 the glycoprotein genes gB,22 gD and gG,13,23 and the thymidine kinase gene.12 Since nucleic acid amplification assays are rapid, sensitive and specific for detecting HSV, a simple one-step duplex PCR test for the diagnosis and genotyping of HSV was developed. The PCR assay targets a specific region of the glycoprotein D (gD) gene, and simultaneously and unambiguously detects and genotypes HSV-1 and 2 infections in CSF specimens. Two pairs of primers specific for HSV-1 and 2 and a specific probe for confirmation of PCR results by Southern blot were designed and used to develop the PCR test. Uracyl-N-glycosylase (UNG) was incorporated in the assay to control and minimize PCR carry-over contamination. To demonstrate the specificity, sensitivity and reproducibility of the assay, a region of the HSV gD gene that included the primer sequences was isolated, cloned and used as a positive control for detecting and estimating the amount of HSV DNA in CSF specimens.

cloning vector (Invitrogen) following the manufacturer’s instructions and using recombinant DNA techniques.25 The recombinant clone was purified with a plasmid kit (Qiagen). The insert, flanked at each end by an EcoRI site, was sequenced (ABI 373XL sequencer) in both directions using the T7 and M13 primers to verify the HSV target sequence.

Selection of primers for PCR and probe for Southern blot The HSV-1 locus HE1CG, Emb database accession number X14112, and HSV-2 locus HSV2HG52, Emb database accession number Z86099, were used to design unique primers that amplify both HSV-1 and 2 target sequences. The primers sense (A), 5′-ATCCGA-ACG-CAG-CCC-CGC-TG and antisense (B): 5′TCC-GTC-CAG-TCG-TTT-ATC-TTC-AC-3′ generate a PCR product of 140 bp. A second sense primer (C): 5′-GGA-CGA-GGC-CCG-AAA-GCA-CAC-G-3′ used in combination with the antisense primer (B) generates a PCR product of 270 bp, and differentiates and genotypes HSV-2. A 23 base 5′-amino-modified oligonucleotide, 5′-NH2-AGC-GCC-GTC-AGC-GAG-GATAAC-CT-3′, was designed and used as a probe in Southern blot for detection of both HSV-1 and 2. The probe was labelled with an alkaline–phosphatase conjugate and detected with a chemiluminescent substrate (Boheringer Mannheim).

DNA purification from controls and clinical samples MATERIALS AND METHODS Selection of target sequences and primers for HSV control The glycoprotein D (gD) gene of the HSV genome was selected as a target for the design of primer for PCR amplification and the preparation of the positive control. A strain of HSV-2 isolated from a patient and cultured in Vero cells,24 was used as the source of DNA. The primers, sense: 5′-GCC-TGC-CGC-AGCGTG-CTC-CTA-3′ and antisense: 5′-GGT-GCT-CCAGGA-TAA-ACT-GTG-T-3′ were used to amplify a 358 bp fragment. The gD gene sequence of the locus HSV2HG52, Emb database accession number Z86099, was used to design these primers. Polymerase chain reaction products were electrophoresed on an ethidium bromide stained agarose gel and purified with a PCR purification kit (Giagen QIAquick) following the manufacturer’s instructions. The purified fragment was cloned into a 3·9 kb PCR 2·1 TA

HSV-1 and 2 DNA were extracted and purified from virus cultured stocks and from 15 CSF specimens from infants (BBI-Clinical Laboratory) using a nucleic acid purification kit (Boheringer Mannheim) consisting of the specific binding and elution of DNA on glass fibers in the presence of a chaotropic salt. The HSV DNA from CSF was also extracted by direct boiling (5 min) in TE buffer25 and centrifugation in a microcentrifuge. The clinical samples were also independently analysed for HSV by BBI-Clinical Laboratory in CT, USA. Aliquots of 5–10 ll of purified DNA were used for PCR.

PCR assay A simple one-step duplex PCR assay was developed to simultaneously amplify HSV-1 & 2. The reaction mixture contains 1× PCR Buffer II (Perkin Elmer), 2 m MgCl2, 150 l dATP, dCTP, dGTP and dUTP

Detection and genotyping of H. simplex

(Boheringer Mannheim), 25 pmoles of the sense primer A and 50 pmoles each of the antisense primer B & the sense primer C, 0·25 U of Uracyl-N-Glycosylase (Epicentre Technologies), 3 U of Taq DNA Polymerase (Amplitaq, Perkin Elmer), 5 ll DNA template and distilled-deionized water to a final volume of 50 ll. Amplification was peformed in a Perkin Elmer 9600 thermocycler and subjected to a cycling profile consisting of incubation at 50°C for 2 min and DNA denaturation at 95°C for 5 min followed by 40 cycles of amplification consisting of a denaturation step at 94°C for 1 min, an annealing step at 58°C for 45 s and an extension step at 72°C for 30 s. A final extension step at 72°C for 10 min and a termination step at 4°C were also included.

Analysis of PCR products All PCR products and their size were evaluated by ethidium bromide stained agarose (2%) gel electrophoresis using a DNA size marker (100 bp ladder) and the HSV clone as a positive control. The PCR products were transferred to nylon membranes by Southern blot hybridization using a HSV-1/2 specific oligonucleotide-alkaline phosphatase (oligo-AP) conjugated probe. The DNA transfer was carried out in 0·4  NaOH. The membrane was rinsed in 2×SSC (Sambrook et al., 1989), u.v. crosslinked at 1200 lJ and hybridized to the probe (BBI-Biotech Research Labs system). After hybridization the membrane was washed with appropriate stringency conditions, and detection was achieved using the Chemiluminescent substrate CSPD (Boheringer Mannheim). After 5 min the membrane was placed in a film cassette, and Kodak XAR film was exposed from 20 min to 2 h at room temperature and developed. This procedure was used to confirm the PCR results.

RESULTS Location of primers, probes and HSV control in the gD gene The position of primers, probes and HSV control sequences in the HSV gD gene is shown in Fig. 1. The primers A and B amplify a highly conserved sequence of the gD gene that is common to both HSV-1 and 2. The primers C and B amplify an HSV2 specific sequence. The oligonucleotide probe is located within these two fragments and harbours a conserved sequence that specifically hybridizes and identifies both HSV-1 and 2. The HSV control, cloned into the TA vector, harbours a 358 bp insert that

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contains the sequences of the primers and the oligonucleotide probe; thus, it can be used as a positive control for both HSV-1 and HSV-2 in PCR and Southern blot hybridization.

Primers specificity and genotyping The primers A and B generate a fragment of 140 bp that is common to both HSV-1 and 2. Thus, they cannot differentiate the two viral strains. In contrast, the primers C and B amplify a fragment of 270 bp that is specific for HSV-2, and thus, they cannot amplify HSV-1 (Fig. 2a). When the primers A, B and C are added to the PCR reaction mixture (duplex PCR), and both viruses are present in the sample, they amplify and differentiate HSV-1 and 2. These primers simultaneously amplify two DNA fragments, 140 bp for HSV-1/2 and 270 bp for HSV-2 (Fig. 2b). Primer competition between the two different size fragments, was eliminated during amplification. This was achieved by using the Primer A at half (25 pMole/ reaction) the concentration of the primers B and C (50 pMole/reaction).

Assay sensitivity and reproducibility Serial dilutions of cloned HSV control and cultured herpes virus were performed to demonstrate the sensitivity of the assay in detecting minute amounts of virus. Figure 3a shows 10-fold serial dilutions (105 to 1 copy) of HSV control using primers A and B. Endpoint dilutions of cultured HSV-1 and 2 were also performed using either primers A and B or B and C in separate PCR assays (Fig. 3b), or simultaneously (A, B and C) in the presence of HSV-1 and HSV control (Fig. 3c). In all cases, the characteristic DNA fragment size (140 or 270 bp) was generated. The reproducibility of the PCR assay was also demonstrated by performing replicate PCR assays containing a variable copy number of cloned HSV control using the duplex PCR (Fig. 3d).

Analysis of clinical specimens Fifteen CSF samples from infants were genotyped using the duplex PCR assay. Replicates of the cloned HSV control and cultured HSV-2 were included as positive controls in the assay. Three samples (20%) out of 15 CSFs were positive for HSV. Two samples (13%) were positive for HSV-2 and one sample (7%) was positive for HSV-1. The PCR fragments were

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–3'

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Fig. 1. Schematic representation of the gD gene of Herpes simplex virus (HSV) showing the position of the polymerase chain reaction (PCR) primers, probe and sequence of the cloned HSV insert used to develop the duplex PCR assay. Primers A and B, and C and B generate a 140 and 270 bp fragment, respectively. The sequence of the HSV control is 358 bp in length and is common to both HSV-1 and 2. (a)

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Fig. 2. Ethidium bromide stained agarose (2%) gel pictures. (a) M=marker (100 bp DNA ladder), 1–5 [replicates of HSV-1 polymerase chain reaction (PCR) products (140 bp) generated with primers A and B], 5 and 10 (negative buffer controls), 6–9 [replicates of HSV-2 PCR products (270 bp) generated with primers C and B]. (b) M (100 bp DNA ladder), 1–4 [replicates of HSV-1 and 2 PCR products (140 bp and 270 bp fragments) amplified with primers A, B and C].

clearly visible in the ethidium bromide stained agarose gel. Southern blot analysis was performed with the PCR products of these samples to confirm the electrophoresis results and ensure that no PCR products invisible to the naked eye could be left undetected in the agarose gel. Hybridization was performed with an aligonucleotide probe, which is highly specific for both HSV-1 and 2. The Southern hybridization assay confirmed the PCR results (Fig. 4). No additional bands were observed. These assays were repeated two additional times. In all cases the same results were obtained. The CSF samples were also analysed by a different laboratory. Perfect concordance was observed between our results and those obtained by the independent laboratory when the code of the samples was broken and results compared.

DISCUSSION The results presented here show a highly specific, sensitive and reproducible duplex PCR assay for the

detection and genotyping of HSV-1 and 2. This robust one-step assay is user-friendly, simple and can be easily implemented in the clinical laboratory for routine diagnosis of HSV infections. Although agarose gel electrophoresis was sufficient to detect as low as 10 copies of HSV (Fig. 2a), the chemiluminescent oligonucleotide probe not only allowed the detection of one copy of HSV (Fig. 3a), but confirmed the gel results and ensured that minute amounts of the viruses were not left undetected. This duplex PCR assay provides several advantages. It can be used in uniplex format by combining the appropriate pair of primers (A and B for detection of HSV-1 and 2 or C and B for detection and differentiation of HSV-2) in separate PCR reactions. The assay can also be used as a screening tool when large numbers of samples need to be tested and no genotyping is required. The primer pair A and B can be used for this purpose. These primers detect both HSV-1 and 2 but cannot differentiate them because the primers generate an equal size fragment (140 bp) and amplify a highly conserved sequence that is present in both viruses. If genotyping

Detection and genotyping of H. simplex (a)

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(c) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 270 bp 140 bp

(d) 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 B 270 bp 140 bp Replicate a

Replicate b

Fig. 3. X-ray film photographs of polymerase chain reaction (PCR) products transferred to nylon membranes (Southern blot) and hybridized to a chemiluminescentlabelled probe. (a) 10-fold serial dilutions of cloned HSV control. The PCR products (140 bp) were generated with primers A and B. (b) Two-fold end-point serial dilutions of cultured HSV-1 (1–7) and HSV-2 (1–9). PCR products were generated with primers A and B for HSV-1 (140 bp) and primers B and C for HSV-2 (270 bp) in separate PCR reactions. (c) Two-fold end-point serial dilutions of cultured HSV-1 (1–9), negative controls (10 and 13), and cultured HSV-1 (11 and 12) and HSV-2 (14) as positive controls. PCR fragments (140 and 270 bp) were generated with primers A, B and C (duplex PCR). (d) Cloned HSV control at different copy numbers. Replicate a: 1 (105), 2 (104), 3 (103), 4 (102), 5–9 (10 copies); Replicate b: 1 (104), 2 (103), 3–8 (102 copies), B (negative buffer control). PCR products (140 and 270 bp) were generated with primers A, B and C (duplex PCR).

is later required, HSV positive samples could then be amplified with the primers C and B to determine the genotype of the virus. Specimens containing HSV-2 will generate a 270 bp fragment, while specimens containing HSV-1 will be negative. One more advantage of this PCR assay is that the reaction mixture has already incorporated UNG. This is essential to prevent carry-over contamination, especially when a large number of samples are screened. An additional advantage of this assay is that it can be scaled down

270 bp 140 bp

Fig. 4. X-ray film photograph of polymerase chain reaction (PCR) products generated from cerebrospinal fluid (CSF) specimens that were transferred to nylon membranes (Southern blot) and hybridized to a chemiluminescent-labelled probe. M=600 bp marker, 1 and 4 (specimens positive for HSV-2), 2 (specimen positive for HSV-1). Numbers 3, and 5 to 12 show Herpes simplex virus (HSV) negative specimens. Specimens 13, 14 and 15 (not shown) were also virus negative.

(25 or 12·5 ll) to economize reagents when high throughput screening is required. Thus the simplicity of this assay makes it an excellent candidate for routine diagnosis of HSV in clinical laboratories. Although the assay provides the flexibility of being performed in uniplex and/or duplex formats, the main purpose of this assay is to genotype CSF specimens from infants using the duplex PCR with the three primers (A, B and C). Infants are particularly at risk of herpes encephalitis. Thus, HSV testing is of epidemiological importance because HSV-2 causes encephalitis and, if left untreated, the infection can lead to neurological sequelae and brain damage. Knowledge of the HSV genotype can help to determine and design appropriate antibiotic therapies for infected patients. The PCR assay is currently used to screen specimens from neonates who have been exposed to HSV during birth and are at risk for developing encephalitis.

ACKNOWLEDGEMENTS The author wishes to thank Dr Richard C. Tilton, of BBIClinical Laboratories, CT, USA, for providing the CSF specimens, and Kezuo Wu, Lijuan Yang and Diana Ngo for their excellent technical assistance.

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