Brian P. Holloway

Real-Time Reverse Transcription-PCR Assay for Comprehensive Detection of Human Rhinoviruses

ABSTRACT Human rhinoviruses (HRVs) are important contributors to respiratory disease, but their healthcare burden remains unclear, primarily because of the lack of sensitive, accurate, and convenient means of determining their causal role. To address this, we developed and clinically validated the sensitivity and specificity of a real-time reverse transcription-PCR (RT-PCR) assay targeting the viral 5′ noncoding region defined by sequences obtained from all 100 currently recognized HRV prototype strains and 85 recently circulating field isolates. The assay successfully amplified all HRVs tested and could reproducibly detect 50 HRV RNA transcript copies, with a dynamic range of over 7 logs. In contrast, a quantified RNA transcript of human enterovirus 68 (HEV68) that showed the greatest sequence homology to the HRV primers and probe set was not detected below a concentration of 5 × 105 copies per reaction. Nucleic acid extracts of 111 coded respiratory specimens that were culture positive for HRV or HEV were tested with the HRV real-time RT-PCR assay and by two independent laboratories that used different in-house HRV/HEV RT-PCR assays. Eighty-seven HRV-culture-positive specimens were correctly identified by the real-time RT-PCR assay, and 4 of the 24 HEV-positive samples were positive for HRV. HRV-specific sequences subsequently were identified in these four specimens, suggesting HRV/HEV coinfection in these patients. The assay was successfully applied in an investigation of a coincidental outbreak of HRV respiratory illness among laboratory staff.

Detection and identification of vaccine-related polioviruses by the polymerase chain reaction

We have used the polymerase chain reaction (PCR) to obtain sensitive detection and identification of poliovirus RNA genomes. Primer pairs were designed to permit identification of each Sabin poliovaccine strain by the electrophoretic mobilities of the amplified DNA products (Sabin 1: 97 bp; Sabin 2: 71 bp; Sabin 3: 44 bp). The compositions of samples containing mixtures of vaccine strains could be readily determined by PCR. When the amplified products were visualized by ethidium bromide fluorescence, as few as 250 genomic copies in the original sample could be detected. When PCR was used in combination with strain-specific 32P-labeled oligonucleotide probes, the limit of detection was less than or equal to 2.5 poliovirus genomes, exceeding the sensitivity of poliovirus isolation in cell culture by at least 100-fold. PCR amplifications may be performed on virion RNAs extracted directly from clinical specimens, potentially eliminating the requirement for virus isolation in routine identifications while yielding reliable results within 8 h.

Real-time reverse transcription-polymerase chain reaction assay for SARS-associated coronavirus.

A real-time reverse transcription–polymerase chain reaction (RT-PCR) assay was developed to rapidly detect the severe acute respiratory syndrome–associated coronavirus (SARS-CoV). The assay, based on multiple primer and probe sets located in different regions of the SARS-CoV genome, could discriminate SARS-CoV from other human and animal coronaviruses with a potential detection limit of <10 genomic copies per reaction. The real-time RT-PCR assay was more sensitive than a conventional RT-PCR assay or culture isolation and proved suitable to detect SARS-CoV in clinical specimens. Application of this assay will aid in diagnosing SARS-CoV infection.

Development and Evaluation of Real-Time PCR-Based Fluorescence Assays for Detection of Chlamydia pneumoniae

ABSTRACT Chlamydia pneumoniae is an important respiratory pathogen recently associated with atherosclerosis and several other chronic diseases. Detection of C. pneumoniae is inconsistent, and standardized PCR assays are needed. Two real-time PCR assays specific for C. pneumoniae were developed by using the fluorescent dye-labeled TaqMan probe-based system. Oligonucleotide primers and probes were designed to target two variable domains of the ompA gene, VD2 and VD4. The limit of detection for each of the two PCR assays was 0.001 inclusion-forming unit. Thirty-nine C. pneumoniae isolates obtained from widely distributed geographical areas were amplified by the VD2 and VD4 assays, producing the expected 108- and 125-bp amplification products, respectively. None of the C. trachomatis serovars, C. psittaci strains, other organisms, or human DNAs tested were amplified. The amplification results of the newly developed assays were compared to the results of culturing and two nested PCR assays, targeting the 16S rRNA and ompA genes. The assays were compared by testing C. pneumoniae purified elementary bodies, animal tissues, 228 peripheral blood mononuclear cell (PBMC) specimens, and 179 oropharyngeal (OP) swab specimens obtained from ischemic stroke patients or matched controls. The real-time VD4 assay and one nested PCR each detected C. pneumoniae in a single, but different, PBMC specimen. Eleven of 179 OP specimens (6.1%) showed evidence of the presence of C. pneumoniae in one or more tests. The real-time VD4 assay detected the most positive results of the five assays. We believe that this real-time PCR assay offers advantages over nested PCR assays and may improve the detection of C. pneumoniae in clinical specimens.

Quantitative, Fluorogenic Probe PCR Assay for Detection of Human Herpesvirus 8 DNA in Clinical Specimens

ABSTRACT A quantitative, fluorescence-based PCR assay (TaqMan-based system) was developed for detection of human herpesvirus 8 (HHV-8) DNA in clinical specimens. Primers and probes chosen from each of five 10-kb segments from the unique region of the HHV-8 genome were evaluated for sensitivity with dilution series of DNA extracted from a cell line (BCBL-1) that harbors HHV-8 DNA. Although several of the primer-probe sets performed similarly with BCBL-1 DNA that had been diluted in water, their performance differed when target DNA was diluted in a constant background of uninfected cell DNA, an environment more relevant to their intended use. The two best primer-probe combinations were specific for HHV-8 relative to the other known human herpesviruses and herpesvirus saimiri, a closely related gammaherpesvirus of nonhuman primates. PCRs included an enzymatic digestion step to eliminate PCR carryover and an exogenous internal positive control that enabled discrimination of false-negative from true-negative reactions. The new assays were compared to conventional PCR assays for clinical specimens (saliva, rectal brushings, rectal swab specimens, peripheral blood lymphocytes, semen, and urine) from human immunodeficiency virus-positive patients with or without Kaposis sarcoma. In all instances, the new assays agreed with each other and with the conventional PCR system. In addition, the quantitative results obtained with the new assays were in good agreement both for duplicate reactions in the same assay and between assays.

Temperature-Mediated Heteroduplex Analysis Performed by Using Denaturing High-Performance Liquid Chromatography To Identify Sequence Polymorphisms in Mycobacterium tuberculosis Complex Organisms

ABSTRACT PCR products containing sequence polymorphisms were prepared from six mycobacterial genes, denatured, mixed with reference PCR products, and reannealed; the mixtures were then examined with a denaturing high-performance liquid chromatography system (WAVE) equipped with a temperature-controlled alkalated polystyrene divinyl benzene column. Mismatching of bases in heteroduplexes of the PCR products causes elution patterns of the DNA from the column to be altered. The six mycobacterial genes studied were oxyR, in which a specific polymorphism (G1031A) is found only in certain species of the Mycobacterium tuberculosis complex, and five genes in which mutations associated with antituberculosis drug resistance have been found. The resistance genes (with affected drug and PCR product sizes given parenthetically) were rpoB (rifampin; 258 bp), katG (isoniazid; 205 bp), pncA (pyrazinamide; 579 bp); rpsL (streptomycin; 196 bp), and embB (ethambutol; 185 bp). Elution patterns of heteroduplexes of all 20 polymorphisms studied shifted detectably at column temperatures ranging from 65.3 to 68°C and elution times of 3.5 to 6 min. These results show that temperature-mediated heteroduplex analysis is a potentially useful genotypic screen for mutations associated with antituberculosis drug resistance and for the G1031A polymorphism in oxyR. The method may allow users to detect novel as well as heterogeneous mutations without using expensive kits or detection labels.

Evaluation of the Invader Assay, a Linear Signal Amplification Method, for Identification of Mutations Associated with Resistance to Rifampin and Isoniazid in Mycobacterium tuberculosis

ABSTRACT We evaluated a recently described linear signal amplification method for sensitivity and specificity in detecting mutations associated with resistance to rifampin (RIF) and isoniazid (INH) inMycobacterium tuberculosis. The assay utilizes the thermostable flap endonuclease Cleavase VIII, derived fromArchaeoglobus fulgidus, which cleaves a structure formed by the hybridization of two overlapping oligonucleotide probes to a target nucleic acid strand. This method, termed the Invader assay, can discriminate single-base differences. Nine pairs of probes, encompassing five mutations in rpoB and katGthat are associated with resistance to either RIF or INH, as well as the corresponding wild-type (drug-susceptible) alleles, were tested using amplified DNA. Fluorescent-labeled cleavage products, ranging from 4 to 13 nucleotides in length, depending on the genotype of the test sample, were separated by denaturing polyacrylamide (20 to 24%) gel electrophoresis and then detected by scanning. All nine alleles could be identified and differentiated on the basis of product size. Multiple mutations at a specific rpoB nucleotide in target PCR products could be identified, as could mutants that were present at ≥0.5% of the total population of target sequences. The Invader assay is a sensitive screen for some mutations associated with antituberculosis drug resistance in amplified gene regions.

The structural proteins of rabies virus and evidence for their synthesis from separate monocistronic RNA species.

Purified preparations of the CVS strain of rabies virus, which were labelled during the infectious growth cycle with different isotopes or labelled in vitro by iodination or acetylation, contained five major proteins, L, G, N, M1, and M2, when examined by polyacrylamide gel electrophoresis (PAGE). The major surface glycoprotein, G, could be separated into two components, G1, G2, in some PAGE systems; they were present in about equimolar amounts and had apparent mol. wt. of 70.5 X 10(3) and 65 X 10(3), respectively. The virus nucleocapsid (p = 1.28 g/ml) could be isolated after detergent treatment of purified virus. It contained the virus RNA, the major nucleocapsid protein, N (mol. wt. 58.5 X 10(3)), and small amounts of a large protein, L (mol. wt. 170 X 10(3)). Two membrane proteins, M1 (mol. wt. 39.5 X 10(3)) and M2 (mol. wt. 25 X 10(3)), were also observed. Chromatography of dissoliated rabies virus in agarose columns with guanidine hydrochloride did not resolve any additional virus structural proteins. Two-dimensional peptide may analysis of iodinated structural proteins indicated that they were unique gene products and not derived from a precursor polypeptide by cleavage. The peptide maps of the two glycoproteins, G1 and G2, appeared identical. The approximate number of proteins molecules per virion has been determined. Rabies virus-directed protein synthesis in BHK21 cultures was detected as early as 6 h p.i. and all the proteins were present 12h p.i. Additional non-structural virus-specific proteins were not observed. The NaCl hypertonic shock procedure, which differentially inhibits polypeptide chain initiation in different classes of mRNAs, was used to ihibit the synthesis of the G and M1 proteins relative to the others selectively. All the rabies virus proteins were synthesized simultaneously following release from hypertonic treatment, suggesting that there are independent polypeptide chain initiation sites for the synthesis of each of the rabies proteins and that each protein is derived via translation of monocistronic mRNA species.

Development, expression, and murine testing of a multistage Plasmodium falciparum malaria vaccine candidate.

A synthetic gene encoding twelve B cell epitopes, six T-cell proliferative epitopes, and three cytotoxic T lymphocyte (CTL) epitopes from nine stage-specific antigens, representing the sporozoite, liver stage, asexual blood-stage, and sexual-stage antigens of Plasmodium falciparum, was constructed by assembling overlapping oligonucleotides followed by PCR extension and annealing. A three-step PCR protocol using twelve long oligonucleotides was employed to generate a 1053 base-pair synthetic gene, the identity of which was confirmed by sequencing. This synthetic gene, named CDC/NII MAL VAC-1, was cloned, and the recombinant protein was expressed in the Baculovirus Expression Vector System (BEVS). The selection of malarial epitopes for inclusion in this vaccine construct was based on immunoepidemiological studies in malaria endemic area, in vitro, and in vivo protection studies in model systems. The 41 kDa BEVS-expressed recombinant protein reacted with mouse antibodies specific for individual B cell epitopes in the vaccine construct and with sera from clinically immune Kenyan adults. An immunization study in three strains of mice that differ at the H-2 locus demonstrated that the BEVS-expressed recombinant protein is immunogenic; the candidate vaccine antigen induced high titer antibodies, and lymphocyte proliferative and IFN-gamma responses. These results demonstrate that individual B and T cell epitopes can be assembled to create synthetic genes that encode proteins capable of eliciting specific antibody and T cell responses.

Rabies virus-induced RNA synthesis in BHK21 cells.

Rabies virus polysomes contained two sizes of messenger RNAs, one of which had a sedimentation value of 30S and another which sedimented at 12 to 16S. RNA extracted from infected cultures contained virion-size RNA, 42S, as well as 30S and 12 to 16S RNA species. Hybridization studies indicated that the 30S and 12 to 16S RNAs had nucleotide sequences which were complementary to virion RNA. RNA. RNA isolated from virus polysomes contained adenylate-rich sequences which were heterogeneous in size and were determined to be about 100 to 250 nucleotides in length on the basis of their migration rates in polyacrylamide gels. Acid-urea agarose gel electrophoresis established that the 30S RNA material was composed of a single RNA species (mol. wt. greater than or equal to 1.65 X 10(6)), whereas the 12 to 16S material could be resolved into at least four distinct species whose mol. wt. ranged from 0.28 to 0.87 X 10(6). When labelled rabies-infected cell RNAs, which were purified by oligo(dT)-cellulose chromatography, were annealed to excess unlabelled virus RNA, digested with ribonuclease T2 and the RNA duplex molecules analysed by polyacrylamide gel electrophoresis, five duplexes could be separated. The mol. wt. of these duplexes were estimated to be 3.2, 1.4, 0.96, 0.55 and 0.39 X 10(6), when compared to the known mol. wt. of vesicular stomatitis virus (VSV) RNA duplexes. This study suggests that the replicative processes of rabies virus are very similar to VSV and that rabies virus proteins are probably translated from smaller than virion-size RNAs.