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PubMed2001

Multiplex allele-specific target amplification based on PCR suppression.

We have developed a strategy for multiplex PCR based on PCR suppression. PCR suppression allows DNA target amplification with only one sequence-specific primer per target and a second primer that is common for all targets. Therefore, an n-plex PCR would require only n + 1 primers. We have demonstrated uniform, efficient amplification of targeted sequences in 14-plex PCR. The high specificity of suppression PCR also provides multiplexed amplification with allele specificity. Multiplexed PCR was used to develop assays for genotyping DNA samples from cystic fibrosis-affected individuals. The new approach greatly simplifies primer design, significantly increases the PCR multiplexing level, and decreases the overall primer cost. In addition, this assay is more readily amenable to automation and is therefore suitable for high-throughput genetic diagnostics.

Alleles↗

PubMed2001

Detection of macrolide resistance mechanisms in Streptococcus pneumoniae and Streptococcus pyogenes using a multiplex rapid cycle PCR with microwell-format probe hybridization.

In this study, a multiplex rapid cycle PCR with microwell-format probe hybridization method was developed to perform high-volume screening for macrolide resistance determinants in isolates of Streptococcus pneumoniae and Streptococcus pyogenes. The method was then utilized to determine the distribution of macrolide resistance mechanisms in recent isolates of S. pneumoniae and S. pyogenes from Great Britain and Ireland. For 83 strains of macrolide resistant S. pneumoniae tested, 51 (61.4%) were positive for mef(A), 29 (34.9%) erm(B), two (2.4%) double mechanisms mef(A) + erm(B), and one (1.2%) negative for all mechanisms tested. For 56 strains of macrolide-resistant S. pyogenes tested, 33 (58.9%) were positive for erm(A) subclass erm(TR), 18 (32.1%) mef(A) and five (8.9%) erm(B).

Anti-Bacterial Agents↗

PubMed2006

Development and evaluation of a multiplex RT-PCR for detecting main viruses and a viroid of potato.

A multiplex RT-PCR (mRT-PCR) for detecting four potato viruses (Potato virus S (PVS), Potato virus X (PVX), Potato virus Y (PVY), and Potato leaf roll virus (PLRV)) and one potato viroid (Potato spindle tuber viroid (PSTVd)) was developed. The mRT-PCR consisted of one reaction with specific primers designed according to the sequences of coat protein (CP) genes of respective viruses and the sequence of the viroid. The entire procedure from tissue grinding to RT-PCR results takes about 4 hrs. The reliability of the method was tested on leaves sampled from the field, greenhouse and in vitro-grown plants by comparison with double-antibody sandwich ELISA (DAS-ELISA). A high correlation between these two methods was observed. The mRT-PCR was also evaluated by testing infected samples obtained from the International Potato Center, Lima, Peru; this testing confirmed its high reliability and sensitivity.

Capsid Proteins↗

PubMed2002

Single nucleotide polymorphisms and microsatellite alleles of tumor necrosis factor alpha and interleukin-10 genes and the risk of advanced chronic alcoholic liver disease.

BACKGROUND: Only a minority of ethanol abusers develop advanced chronic alcoholic liver disease (CALD). In CALD there is a imbalance between TNF alpha and IL-10, which may be modulated by several polymorphisms at both genetic loci. Our aim has been to elucidate the possible relation between these polymorphisms and the risk of CALD. PATIENTS AND METHODS: 147 patients with advanced CALD and 355 healthy controls (all white Spaniards) were included. TNF alpha biallelic single nucleotide polymorphisms (SNP) at positions -238, -308, and -376 and IL-10 biallelic SNP at positions -597, - 824, and - 1087 were investigated by polymerase chain reaction (PCR) amplification and dot blot hybridization. Moreover, polymorphic microsatellites TNFa, TNFb, IL-10.R and IL-10.G were investigated in a multiplex PCR and alleles were estimated in an automatic sequencer. RESULTS: No differences were found in the distribution of any of the studied polymorphisms, except by an excess of the haplotype formed by the allele 11 of the microsatellite IL-10.G and the GCC arrangement of the SNPs at the promoter of IL-10 gene in patients (15.7 vs. 8.24%, odds ratio: 2.08, 95% C.I. = 1.31-3.31). CONCLUSIONS: The studied polymorphisms at TNF alpha and IL-10 genetic loci are not clearly related to the risk of CALD. The excess of G11-GCC haplotype found in CALD patients needs independent confirmation.

Adult↗

PubMed2006

Isolation and molecular biological investigations of avian poxviruses from chickens, a turkey, and a pigeon in Croatia.

In the last 3 yr, several outbreaks of avian poxviruses (APVs) have been observed in different parts of Croatia. Four strains of APVs, from chickens, a pigeon, and a turkey, were isolated from cutaneous lesions by inoculation onto the chorioallantoic membranes (CAM) of 12-day-old specific-pathogen-free chicken embryos. The resulting proliferative CAM lesions contained eosinophilic cytoplasmic inclusion bodies. The characteristic viral particles of poxvirus were detected in the infected CAM and also in the infected tissues by transmission electron microscopy. Further identification and differentiation of the four various APVs were carried out by the use of a polymerase chain reaction (PCR) combined with restriction enzyme analysis. Using one primer set, which framed a region within the APV 4b core protein gene, it was possible to detect APV-specific DNA from all four tested isolates. PCR results revealed no recognizable differences in size of amplified fragments between the different APVs from chickens, turkey, and pigeon. Restriction enzyme analysis of PCR products using NlaIII showed the same cleavage pattern for turkey and chicken isolates and a different one for the pigeon isolate. Multiplex PCR for direct detection of APV and reticuloendotheliosis virus (REV) was carried out to determine the possible integration of REV in the genome of isolated APVs. The obtained results revealed that REV was present in chicken and turkey strains of poxviruses, whereas the pigeon isolate was negative. It is not known whether the avipoxvirus vaccine strain used in Croatia is contaminated with REV or if the REV is naturally contaminating Croatian field strains of fowl poxvirus. The latter is indicated by the negative REV finding in the pigeon, which was not vaccinated. The results of the present study indicate the reemergence of fowlpox in Croatia, where infections have not been recorded since 1963 and never confirmed etiologically.

Animals↗

PubMed2001

Quantitation of marrow disease in neuroblastoma by real-time reverse transcription-PCR.

PURPOSE: GD2 is abundantly expressed in neuroblastoma (NB). GD2 synthesis is dependent on key enzyme beta 1,4-N-acetylgalactosaminyltransferase (GD2 synthase). We explore the potential of GD2 synthase mRNA as a molecular marker of minimal residual disease by first comparing it quantitatively with immunocytology and then testing its clinical utility. EXPERIMENTAL DESIGN: A real-time reverse transcription-PCR assay to quantify mRNA of GD2 synthase was developed. Quantitation was normalized to endogenous control glyceraldehyde-3-phosphate dehydrogenase in a multiplex PCR. RESULTS: The upper limit of normal was defined by 31 normal marrow and blood samples, achieving a sensitivity of one NB cell in 10(6) normal mononuclear cells. When 155 bone marrows from 100 NB patients were studied, GD2 synthase mRNA levels correlated well with the number of GD2-positive cells, as measured by immunocytology using anti-GD2 antibodies (r = 0.96). This is the first demonstration of the quantitative relationship between a specific mRNA and the actual number of tumor cells. In a pilot study, the level of this transcript in sequential marrow samples of five stage 4 NB patients correlated closely with their clinical status. At 24 months after diagnosis, available remission bone marrows from patients with advanced NB diagnosed at >1 year of age initially treated with protocols N6 and N7 at Memorial Sloan-Kettering Cancer Center (n = 44) were analyzed for GD2 synthase mRNA. Positivity was strongly associated with progression-free (P < 0.005) and overall survival (P < 0.001). CONCLUSIONS: Measurement of tumor cells by real-time quantitative reverse transcription-PCR of GD2 synthase has potential clinical utility, especially for the detection of minimal residual disease.

Bone Marrow Cells↗

PubMed2000

Detection and identification of human parainfluenza viruses 1, 2, 3, and 4 in clinical samples of pediatric patients by multiplex reverse transcription-PCR.

We describe a multiplex reverse transcription-PCR (m-RT-PCR) assay that is able to detect and differentiate all known human parainfluenza viruses (HPIVs). Serial dilution experiments with reference strains that compared cell culture isolation and m-RT-PCR showed sensitivities ranging from 0.0004 50% tissue culture infective dose (TCID(50)) for HPIV type 4B (HPIV-4B) to 32 TCID(50)s for HPIV-3. As few as 10 plasmids containing HPIV PCR products could be detected in all cases. When 201 nasopharyngeal aspirate specimens from pediatric patients hospitalized for lower respiratory illness were tested, m-RT-PCR assay detected 64 HPIVs (24 HPIV-3, 23 HPIV-1, 10 HPIV-4, and 7 HPIV-2), while only 42 of them (21 HPIV-1, 14 HPIV-3, 6 HPIV-2, and 1 HPIV-4 isolates) grew in cell culture. Our m-RT-PCR assay was more sensitive than either cell culture isolation or indirect immunofluorescence with monoclonal antibodies for the detection of HPIV infections. Also, HPIV-4 was more frequently detected than HPIV-2 in this study, suggesting that it may have been underestimated as a lower respiratory tract pathogen because of the insensitivity of cell culture.

Animals↗

PubMed1998

Rearranged immunoglobulin light chain genes as minimal residual disease markers in intermediate- and high-grade malignant B cell non-Hodgkin's lymphoma.

Minimal residual disease (MRD) detection in B cell non-Hodgkin's lymphoma (NHL) patients has been shown to be possible using the rearranged heavy (IgH) chain gene as a tumor marker. To explore a second independent tumor marker, we used specific PCR primer sets to identify tumor-specific rearranged Ig light chain (IgL) genes. Rearranged IgL genes were amplified from lymphoma DNA by multiplex PCR using separate primer sets for the Igkappa and the Iglambda genes. They were considered to be of tumor origin if they were monoclonal, and if the same rearrangement was isolated from at least two independent PCR products. From 12 out of 13 intermediate- and high-grade malignant NHL, PCR products could be obtained with IgL specific primers. PCR products from five NHL were studied in detail by cloning and sequencing. The rearranged IgL genes showed 85-100% homology with their closest germ line counterparts. Intraclonal IgL sequence heterogeneity was studied in five lymphomas and detected in only one. Minimal disease was studied in three patients by PCR, followed by Southern hybridization of the PCR product with a lymphoma-specific oligonucleotide probe, which allowed for detection of lymphoma DNA following 1000-fold dilution. Blood samples from one patient, who is in long-term clinical remission, were negative for the lymphoma-specific rearranged Igkappa gene. In the second patient the rearranged Iglambda gene was detected during the first clinical remission, that was followed by a nodal relapse, but not during the second remission, that has been stable for almost 3 years now. The third patient was negative for the rearranged Iglambda gene in blood samples up to 102 months after diagnosis. Circulating lymphoma cells were detected in blood and bone marrow samples which were negative by morphological and immunological criteria. Our studies show that the rearranged IgL gene can be used as a second independent tumor marker in intermediate- and high-grade malignant NHL.

Adult↗

PubMed2002

Evaluation of a commercial diagnostic PCR for the identification of Campylobacter jejuni and Campylobacter coli.

AIMS: DuPont Qualicon recently developed a new PCR assay for the identification of Campylobacter jejuni and Campylobacter coli. We evaluated the selectivity and utility of this assay compared with a PCR method already in use in our laboratory. METHODS AND RESULTS: A group of 133 Campylobacter isolates from poultry carcass rinse samples were screened using the commercial PCR and standard PCR. Identical results were found for 89.5% (119/133) of the isolates. However, 10.5% (14/133) gave conflicting results suggesting mixed cultures. These 14 strains were retested by both PCR methods. Of these, 78.6% (11/14) showed identical results for both PCR methods after retesting; the results for the remaining 21.4% (3/14) again indicated mixed cultures. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF STUDY: The new multiplex PCR is a rapid and accurate alternative to more conventional PCR methods. The persistence of mixed Campylobacter cultures noted in this study suggests certain strains may be very difficult to isolate clonally by standard culture methods.

Animals↗

PubMed2001

Frequent p16/MTS1 inactivation in early stages of urothelial carcinoma of the bladder is not associated with tumor recurrence.

OBJECTIVE: p16, located at chromosome 9p21, is a negative regulator of G1 cell checkpoint and functions as tumor suppressor gene. Only few data are available on the frequency and clinical relevance of p16 alterations in Ta, T1 transitional cell carcinoma (TCC) of the bladder. We investigated 40 patients with Ta, T1 TCC of the bladder for p16 alterations (mutations, homozygote deletions, allelic loss) or reduced p16 immunoreaction. PATIENTS AND METHODS: DNA was prepared from microdissected tumor tissue from 40 patients with pTa, pT1 TCC of the bladder (pTa: 18 patients; pT1: 22 patients; grade 1: 7 patients; grade 2: 28 patients; grade 3: 5 patients). Mutation screening was performed using polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP) and direct sequencing at exon 1 and exon 2. Detection of homozygote deletions was performed using multiplex PCR. Immunohistochemistry (IHC) was performed using an anti-human monoclonal antibody (p16, Pharmingen). Allelic loss was detected by PCR using three different microsatellite markers (D9S161, D9S171, D9S319). RESULTS: SSCP and direct sequencing revealed 3 cases of base substitution which turned out to be natural polymorphisms. Homozygote deletions were not detected in any case. p16 IHC revealed reduced p16 expression (<5% positive nuclei) in 10 patients; 30 patients had a positive reaction (> or =5% positive nuclei) and 10 patients a strong positive reaction (> or =50% positive nuclei). Thirteen of 37 informative cases revealed loss of heterozygosity (LOH) with at least one marker. After a median follow-up of 23 months, 15 patients suffered from disease recurrence. Statistical analysis using Kaplan-Meier analysis and the log-rank test did not reveal significant association of recurrence-free interval and detection of LOH (p = 0.34) or p16 IHC (p = 0.9). CONCLUSIONS: We present a comprehensive evaluation of chromosome 9p21 alterations including p16 analysis and clinical follow-up data. Although p16 mutations and homozygote deletions are rarely detectable in Ta, T1 TCC, the reduction of p16 expression and the frequent hemizygote deletions at 9p21 suggest an early involvement of chromosome 9p and p16 in superficial TCC.

Carcinoma, Transitional Cell↗

PubMed2003

Polymerase chain reaction-based analysis to detect terrestrial animal protein in fish meal.

The recent European bovine spongiform encephalopathy crisis has focused attention on the importance of adopting stringent control measures to avoid the risk of the diffusion of mad cow disease through meat meal-based animal feedstuffs. Potential adulteration of such feedstuffs with bone particles from terrestrial animals is determined by microscopic examination by law before the release of these feedstuffs for free circulation in the European Community. This study describes a DNA monitoring method to examine fish meal for contamination with mammalian and poultry products. A polymerase chain reaction (PCR) method based on the nucleotide sequence variation in the 12S ribosomal RNA gene of mitochondrial DNA was developed and evaluated. Three species-specific primer pairs were designed for the identification of ruminant, pig, and poultry DNA. The specificity of the primers used in the PCR was tested by comparison with DNA samples for several vertebrate species and confirmed. The PCR specifically detected mammalian and poultry adulteration in fish meals containing 0.125% beef, 0.125% sheep, 0.125% pig, 0.125% chicken, and 0.5% goat. A multiplex PCR assay for ruminant and pig adulteration was optimized and had a detection limit of 0.25%.

Animal Feed↗

PubMed1998

Susceptibility to esophageal cancer and genetic polymorphisms in glutathione S-transferases T1, P1, and M1 and cytochrome P450 2E1.

Genetic polymorphisms in enzymes involved in carcinogen metabolism have been shown to influence susceptibility to cancer. Cytochrome P450 2E1 (CYP2E1) is primarily responsible for the bioactivation of many low molecular weight carcinogens, including certain nitrosamines, whereas glutathione S-transferases (GSTs) are involved in detoxifying many other carcinogenic electrophiles. Esophageal cancer, which is prevalent in China, is hypothesized to be related to environmental nitrosamine exposure. Thus, we conducted a pilot case-control study to examine the association between CYP2E1, GSTM1, GSTT1, and GSTP1 genetic polymorphisms and esophageal cancer susceptibility. DNA samples were isolated from surgically removed esophageal tissues or scraped esophageal epithelium from cases with cancer (n = 45), cases with severe epithelial hyperplasia (n = 45), and normal controls (n = 46) from a high-risk area, Linxian County, China. RFLPs in the CYP2E1 and the GSTP1 genes were determined by PCR amplification followed by digestion with RsaI or DraI and Alw26I, respectively. Deletion of the GSTM1 and GSTT1 genes was examined by a multiplex PCR. The CYP2E1 polymorphism detected by RsaI was significantly different between controls (56%) and cases with cancer (20%) or severe epithelial hyperplasia (17%; P < 0.001). Persons without the RsaI variant alleles had more than a 4-6-fold risk of developing severe epithelial hyperplasia (adjusted odds ratio, 6.0; 95% confidence interval, 2.3-16.0) and cancer (adjusted odds ratio, 4.8; 95% confidence interval, 1.8-12.4). Polymorphisms in the GSTs were not associated with increased esophageal cancer risk. These results indicate that CYP2E1 may be a genetic susceptibility factor involved in the early events leading to the development of esophageal cancer.

Adult↗

PubMed2006

Development of multiplex PCRs for evolutionary and forensic applications of 37 human Y chromosome SNPs.

This work describes an efficient and rapid test for typing 37 single nucleotide polymorphisms (SNPs) of the non-recombining region of Y chromosome (NRY) from a minimal amount of DNA using six PCR multiplexes. Markers were drawn following a hierarchical strategy based on the phylogenetic tree of Y chromosome proposed by the Y Chromosome Consortium [The Y Chromosome Consortium, A nomenclature system for the tree of human Y-chromosomal binary haplogroups, Genome Res. 12 (2002) 339-348]. Two multiplexes--arbitrarily named MY1 and MY2--were developed to explore the basal branches of the tree encompassing all the major clades A-R: MY1 for markers M35, M89, M172, M170, M9, M173, M45 and MY2 for markers M52, M216, M174, M181, M201, M91, M96, M214. Four multiplexes able of typing the more superficial branches typical of most frequent European haplogroups E3b, J2, R1 and I, were also developed and named MY-E3b (M78, M107, M224, M165, M148, M81), MY-J2 (M158, M68, M47, M102, M137, M67), MY-R1 (M17, M269, M18, P25, SRY10831.2) and MY-I (M72, M223, M26, M21, M161). SNP genotyping was carried out by hot-start PCR amplification with primers yielding fragments between 63 and 210 nucleotides, followed by minisequencing reaction based on dideoxy single-base extension and capillary electrophoresis of extension products. The sequential application of these multiplexes is a robust and effective resource for typing the most frequent European Y-SNP haplogroups, and appears to be suitable for forensic purposes and evolutionary studies.

Chromosomes, Human, Y↗

PubMed2006

Rapid detection of three large novel deletions of the aspartoacylase gene in non-Jewish patients with Canavan disease.

Canavan disease (CD), an autosomal recessive neurodegenerative disorder, is caused by mutations in the aspartoacylase (ASPA) gene. In the present study, the ASPA gene was analyzed in 24 non-Jewish patients with CD from 23 unrelated families. Within this cohort, we found three large novel deletions of approximate 92, 56, and 12.13 kb in length, using both self-ligation of restriction endonuclease-digested DNA fragments with long-distance inverse PCR and multiplex dosage quantitative PCR analysis of genomic DNA. The 92 kb large deletion results in complete absence of the ASPA gene in one homozygous and one compound heterozygous patient, respectively. The 56 kb large deletion causes absence of the majority of the ASPA gene except for exon 1 alone in a compound heterozygous patient. The 12.13 kb deletion involves deletion of the ASPA gene from intron 3 to intron 5 including exons 4 and 5 (I3 to E4E5I5) in a compound heterozygous patient. Patients with the three large deletions clinically manifested severe symptoms at birth, including seizures. Our study showed that the combined use of long-distance inverse PCR and multiplex dosage quantitative PCR analysis of genomic DNA is a helpful and rapid technique to search for large deletions, particularly for detection of large deletions in compound heterozygous patients.

Amidohydrolases↗

PubMed2001

Rapid and accurate typing of Dichelobacter nodosus using PCR amplification and reverse dot-blot hybridisation.

Here we describe an approach to genotyping D. nodosus, based on variation in the fimbrial subunit gene (fimA), which uses polymerase chain reaction (PCR) amplification and hybridisation to immobilised oligonucleotides (PCR/oligotyping). The variable region of D. nodosus fimA, amplified and labelled with digoxigenin (DIG) in a single multiplex PCR amplification, was hybridised to a panel of group- and type-specific poly-dT tailed oligonucleotides that were immobilised on a nylon membrane strip. A mixture of positive control poly-dT tailed oligonucleotides was also included on the membrane. After hybridisation the membrane was washed to a defined specificity, and DIG-labelled fragments hybridising were detected with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (SCIP). The specificity of the oligonucleotides was verified by the lack of cross-reactivity with D. nodosus fimA sequences that had a single base difference. DNA from 14 footrot samples previously genotyped by PCR-SSCP/sequencing [Vet. Microbiol. 71 (2000) 113], was assayed using the PCR/oligotyping technique. All types of D. nodosus which had been detected previously with a PCR-SSCP/sequencing method were detected by this procedure. However, for three of the 14 footrot samples, PCR/oligotyping detected additional types of D. nodosus. Further PCR amplification using type-specific primers, confirmed that these types of the bacterium were present in the footrot samples. These results indicate that PCR/oligotyping is a specific, accurate, and useful tool for typing footrot samples. In combination with a rapid DNA extraction protocol, D. nodosus strains present in a footrot sample can be accurately identified in less than 2 days.

Amino Acid Sequence↗

PubMed1998

Rapid characterization of mutations in amplified human hprt cDNA by polyacrylamide gel electrophoresis.

Compiling hprt mutation spectra involves the isolation and analysis of numerous 6-thioguanine-resistant clones for identifying characteristic point mutations. Since cDNA amplificates are compulsory intermediates in most mutant classification protocols, we suggest their preliminary characterization by polyacrylamide gel electrophoresis for the rapid distinction of clonal and independent mutants and for streamlining mutant analysis procedures. Based on the human hprt cDNA sequence a strategy was developed for mapping missing exons by analytical digests with a small panel of restriction enzymes. In mutant classification schemes, polyacrylamide gel electrophoresis of AluI-digested cDNA amplificates increased the sensitivity for detecting RT-PCR products of reduced size, e.g., in the case of missing exon 5. Restriction analysis of cDNA amplificates from 109 independent mutant clones showed a significant increase of exon loss after NNK induction as compared to spontaneous or BaP-induced mutants. The determination of exon loss from cDNA amplificates, as carried out for 39 independent mutant PCR products, might direct towards the genomic target sequences carrying the point mutations, that caused the aberrant splicing, thus eliminating the need of laborious multiplex PCR comprising all exons. For single-strand conformation polymorphism (SSCP) analysis of five known point mutations, sub-amplificates comprising exons 7 and 8 of hprt cDNA were obtained. After a combined heat and alkali denaturation of the double-stranded PCR products, the samples were separated in pre-cast polyacrylamide gels under non-denaturing conditions. Five known nucleotide substitutions within the amplified region, including the C508T hot spot mutation, resulted in mobility shifts of single-strand bands relative to the wild type pattern.

Cell Line↗

PubMed2000

Secretor genotyping for A385T, G428A, C571T, C628T, 685delTGG, G849A, and other mutations from a single PCR.

BACKGROUND: The secretor status of an individual is important for disease relationship studies, because it determines the presence of ABH blood group antigens in the gastrointestinal tract and bodily secretions. Routine serologic methods for determining secretor status are unreliable. Current strategies based on PCR for genotyping require relatively large amounts of DNA and have to be done as several separate experiments. STUDY DESIGN AND METHODS: A single, multiplex PCR technique followed by RFLP digestion with four restriction enzymes produced unique genotype profiles for most known secretor (FUT2) mutations. RESULTS: Samples from a range of individuals with common and rare secretor genotypes were analyzed. Each gave unique patterns that allowed secretor genotypes to be determined. CONCLUSION: By using the method described here and genomic DNA, a secretor genotype based on the FUT2 mutations A385T, G428A, C571T, C628T, 685delTGG, and G849A could be accurately determined.

ABO Blood-Group System↗

PubMed2007

Cost-effective real-time reverse transcriptase PCR (RT-PCR) to screen for Dengue virus followed by rapid single-tube multiplex RT-PCR for serotyping of the virus.

Virus detection methodology provides detection of dengue virus in the early phase of the disease. PCR, targeting cDNA derived from viral RNA, has been used as a laboratory-based molecular tool for the detection of Dengue virus. We report the development and use of three real-time one-step reverse transcriptase PCR (RT-PCR) assays to detect dengue cases and serotype the virus involved. The first RT-PCR assay uses SYBR green I as the reporting dye for the purpose of cost-effective screening for dengue virus. The detection limit of the SYBR green I assay was 10 PFU/ml (0.01 equivalent PFU per assay) for all four dengue virus serotypes. The second RT-PCR assay is a duplex fluorogenic probe-based real-time RT-PCR for serotyping clinical samples for dengue viruses. The detection threshold of the probe-based RT-PCR format was 0.1 PFU for serotypes Dengue-1 and Dengue-2, 1 PFU for serotype Dengue-3, and 0.01 PFU for serotype Dengue-4. The third is a fourplex assay that detects any of the four serotypes in a single closed tube with comparable sensitivity. Validation of the assays with local clinical samples collected from 2004 to 2006 revealed that there was an 88% positive correlation between virus isolation and RT-PCR with regard to dengue virus detection and a 100% correlation with seroconversion in subsequent samples. The serotyping results derived from duplex and fourplex assays agree fully with each other and with that derived from immunofluorescence assays.

Aedes↗