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PubMed2001

High-throughput AFLP analysis using infrared dye-labeled primers and an automated DNA sequencer.

Amplified fragment length polymorphism (AFLP) analysis is currently the most powerful and efficient technique for the generation of large numbers of anonymous DNA markers in plant and animal genomes. We have developed a protocol for high-throughput AFLP analysis that allows up to 70,000 polymorphic marker genotype determinations per week on a single automated DNA sequencer. This throughput is based on multiplexed PCR amplification of AFLP fragments using two different infrared dyelabeled primer combinations. The multiplexed AFLPs are resolved on a two-dye, model 4200 LI-COR automated DNA sequencer, and the digital images are scored using semi-automated scoring software specifically designed for complex AFLP banding patterns (AFLP-Quantar). Throughput is enhanced by using high-quality genomic DNA templates obtained by a 96-well DNA isolation procedure.

Electrophoresis↗

PubMed2006

Interactive effect of genetic polymorphism of glutathione S-transferase M1 and smoking on squamous cell lung cancer risk in Korea.

To evaluate the role of the genetic polymorphisms of CYP2E1, GSTM1 and GSTT1, and their interaction with smoking in lung cancer development in Korean males, a hospital-based case-control study was conducted. Histologically confirmed male lung cancer patients (n=171) and male patients with no present or previous history of systemic illness who visited the urology department (n=196) were recruited from Seoul National University Hospital, Korea (1998-1999). CYP2E1 genotypes were determined by PCR-RFLP using RsaI digestion and GSTM1 and T1 genotypes were determined by multiplex PCR. Risks were estimated as odds ratios (ORs) and 95% confidence intervals (CIs) using a logistic regression model adjusting for age and pack-year. Smoking was a significant risk factor for lung cancer (P<0.001). Although genetic polymorphisms of CYP2E1, GSTM1 and T1 were not associated with the overall risk of lung cancer, the GSTM1 null genotype significantly increased the risk of squamous cell lung cancer (OR=1.9, 95% CI=1.04-3.60). An interactive effect between the GSTM1 null genotype and smoking was observed (P=0.04). These results suggest that the GSTM1 null genotype is associated with squamous cell lung cancer and modifies the effect of smoking on squamous cell lung cancer development in Korean males.

Adult↗

PubMed2004

Human genomic DNA analysis using a semi-automated sample preparation, amplification, and electrophoresis separation platform.

The growing importance of analyzing the human genome to detect hereditary and infectious diseases associated with specific DNA sequences has motivated us to develop automated devices to integrate sample preparation, real-time PCR, and microchannel electrophoresis (MCE). In this report, we present results from an optimized compact system capable of processing a raw sample of blood, extracting the DNA, and performing a multiplexed PCR reaction. Finally, an innovative electrophoretic separation was performed on the post-PCR products using a unique MCE system. The sample preparation system extracted and lysed white blood cells (WBC) from whole blood, producing DNA of sufficient quantity and quality for a polymerase chain reaction (PCR). Separation of multiple amplicons was achieved in a microfabricated channel 30 microm x 100 microm in cross section and 85 mm in length filled with a replaceable methyl cellulose matrix operated under denaturing conditions at 50 degrees C. By incorporating fluorescent-labeled primers in the PCR, the amplicons were identified by a two-color (multiplexed) fluorescence detection system. Two base-pair resolution of single-stranded DNA (PCR products) was achieved. We believe that this integrated system provides a unique solution for DNA analysis.

Automation↗

PubMed2007

Development of a multiplex RT-PCR for the simultaneous detection of three viruses of the honeybee (Apis mellifera L.): acute bee paralysis virus, Black queen cell virus and Sacbrood virus.

A single-step multiple-target (multiplex) reverse transcription-PCR (RT-PCR) was developed for the simultaneous detection and differentiation of three economically important viruses of the honeybee Apis mellifera L.: Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV) and Sacbrood virus (SBV). Three compatible sets of primers, specific for each virus, were designed in conserved regions of the viral genomes for use in a one-step (single tube) RT-PCR assay. The individual RT-PCR assays and the combined multiplex assay were optimized for highest sensitivity and specificity. The multiplex RT-PCR assay was tested on field samples collected from Austrian honeybee colonies. All three viruses were detected, and their identity was confirmed by sequencing of the PCR products. The described multiplex RT-PCR proved to be an accurate tool for rapid simultaneous detection of ABPV, BQCV and SBV directly in honeybee specimens.

Animals↗

PubMed2000

Rapid molecular epidemiologic studies of human parainfluenza viruses based on direct sequencing of amplified DNA from a multiplex RT-PCR assay.

Sequencing studies of limited regions of the human parainfluenza viruses (HPIVs) genomes have helped describe patterns of virus circulation and characterize institutional outbreaks of HPIVs-associated respiratory illness. In this study, we sequenced reverse transcription polymerase chain reaction (RT-PCR)-amplified HPIVs RNA obtained from a multiplex RT-PCR assay described previously for simultaneous detection of HPIV-1, 2 and 3. Differences in the nucleotide sequences of limited regions of the HN gene allowed us to distinguish temporally and geographically diverse HPIV isolates (43 HPIV-1, 7 HPIV-2, 12 HPIV-3 isolates from this and previously published studies). In addition, an outbreak of HPIV-3-associated illness among infants on a pediatric ward was investigated by comparing sequences of three ward isolates with three matched community controls. Sequences of all ward isolates were identical and differed from those of the community controls, suggesting a single introduction and nosocomial transmission of the virus. Combining multiplex reverse transcription polymerase chain reaction (RT-PCR) assays with direct sequencing of the PCR products can provide an integrated system for rapid diagnosis and characterization of HPIVs.

Child↗

PubMed1998

Classification and identification of propionibacteria based on ribosomal RNA genes and PCR.

A rapid method was developed to differentiate the genus Propionibacterium from other genera by using a modified multiplex-PCR (MPCR) approach. Three 16S rRNA-targeted oligonucleotide primers were designed to amplify simultaneously two DNA-fragments in the MPCR assay. The universal primer pair bak11w and bak4 (corresponding to the E. coli 16S rRNA positions 8-25 and 1522-1540, respectively) was used in combination with the primer pair bak4 and gd1 (5'-TGCTTTCGATACGGGTTGAC-3'). The later sequence corresponding to a 16S rRNA motif that is unique for the genus Propionibacterium. Propionibacteria were identified by the amplification of a Propionibacterium-genus specific 900-bp fragment whereas MPCR with DNA from other bacteria generated only a DNA fragment of 1500 bp in amplifications with the two universal primers. The whole procedure including cell lysis, MPCR amplification and analysis can be performed within 1 day, detection limits are at approximately 10(3) cfu propionibacteria (or 35 pg DNA). In addition, the taxonomic situation of the genus Propionibacterium was reexamined using a cycle sequencing strategy. Based on the 16S rDNA, a phylogenetic tree of all the Propionibacterium type strains was reconstructed.

Base Sequence↗

PubMed1996

Simultaneous PCR detection of Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus types 1 and 2 from genital ulcers.

A multiplex PCR (M-PCR) assay with colorimetric detection was devised for the simultaneous amplification of DNA targets from Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus (HSV) types 1 and 2. By using target-specific oligonucleotides in a microwell format, 298 genital ulcer swab specimens collected in New Orleans during three intervals from 1992 through 1994 were evaluated. The results of the M-PCR assay were compared with the results of dark-field microscopy and H. ducreyi culture on two different culture media. HSV culture results were available for 99 specimens collected during the third interval. Confirmatory PCR assays targeting different gene sequences for each of the three organisms were used to validate the M-PCR results. Specimens were resolved as positive for the determination of sensitivity if the reference diagnostic test was positive or if the results of both the M-PCR and the confirmatory PCR were positive. The resolved sensitivities of M-PCR for HSV, H. ducreyi, and T. pallidum were 100, 98.4, and 91%, respectively. The resolved sensitivities of HSV culture, H. ducreyi culture, and dark-field microscopy were 71.8, 74.2, and 81%, respectively. These results indicate that the M-PCR assay is more sensitive than standard diagnostic tests for the detection of HSV, H. ducreyi, and T. pallidum from genital ulcers.

Bacteriological Techniques↗

PubMed2003

Nested multiplex RT-PCR for detection and differentiation of West Nile virus and eastern equine encephalomyelitis virus in brain tissues.

A traditional nested reverse transcription-polymerase chain reaction (RT-PCR) assay specific for eastern equine encephalomyelitis (EEE) virus was designed to multiplex with a previously described West Nile (WN) virus nested RT-PCR assay. Differentiation of EEE and WN was based on base pair size of the amplified product. One hundred fifty-seven mammalian and avian brain tissues were tested by EEE/WN nested multiplex RT-PCR, EEE nested RT-PCR, and WN nested RT-PCR, and results were compared with other diagnostic test results from the same animals. Serological and virus isolation testing confirmed the results of the multiplex PCR assay. When compared with cell culture virus isolation, the multiplex assay was shown to be more sensitive in detecting the presence of EEE or WN virus in brain tissues. The multiplex assay was shown to be sensitive and specific for North American EEE and WN and provided a rapid means of identifying both viruses in brain tissues. No apparent sacrifice in sensitivity was observed in the multiplex procedure compared with the individual EEE and WN nested RT-PCR assays. Data collected from an additional 485 multiplex RT-PCR tests conducted during the summer and fall of 2002 further support the validity of the procedure.

Animals↗

PubMed2006

Noninvasive identification of interindividual variation in xenobiotic-metabolizing enzymes: implications for cancer epidemiology and biomarker studies.

In this study, DNA extracted from frozen urine was used in the analysis of polymorphisms in genes coding for xenobiotic-metabolizing enzymes (XMEs). These included single-nucleotide polymorphisms (SNP) in microsomal epoxide hydrolase (mEH), that is, substitutions of tyrosine by histidine in codon 113 (Y113H) and histidine by arginine in codon 139 (H139R), and deletion polymorphisms in glutathione S-transferase (GST) M1 and T1 genes. The concentration of DNA extracted from urine of a Ghanaian population (n = 91) exposed to aflatoxins in their diet ranged from 82.5 to 573 ng/ml urine. Polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) procedures were used for the characterization of mEH polymorphisms, whereas a multiplex PCR method was utilized to identify GST deletion polymorphisms. In total, 91% and 94% of 91 samples were genotyped for mEH exon 3 and exon 4 polymorphisms, respectively. In the multiplex analysis of GST polymorphisms, 94% and 91% of 91 individuals were genotyped for GSTM1 and GSTT1 polymorphisms, respectively. The polymorphisms in the mEH exon 4, GSTM1 and GSTT1, were not in Hardy-Weinberg equilibrium (HWE) except for mEH exon 3. Representative genotypes identified by PCR-RFLP were cloned and sequenced, then confirmed by comparison with reference sequences of human DNA published in the GenBank BLAST database. These results demonstrate that XMEs can be genotyped from urine with reliable accuracy and may be useful in cancer and molecular epidemiology studies.

Adult↗

PubMed1999

[Detection of RB1 mutations by using quantitative fluorescent mutiplex PCR].

OBJECTIVE: To develop a half-automatic, simple, non-radioactive technique for rapid detection of mutation in the RB gene. METHODS: Quantitative fluorescent multiplex PCR (QFM-PCR) involves amplification of the promoter region and all 27 exons of the RB1 gene with fluorescein labeled primers in multiplex sets. Primers were divided into multiplex sets of three to seven pairs of primers, also contained were internal control primers C4. Four external controls were also tested by using nullisomic, monosomic, diploid, and trisomic for RB1. The number of copies of a fragment in a test sample was calculated by comparing fluorescence intensity of the fragment with these standards. Fragment value detection and subsequent calculations were performed automatically by Fragment Manager 2.1 Software. RESULTS: Small deletions, insertions and whole exon deletion in the RB1 gene were detected from genomic DNA. Sequencing analysis and RB tumor homozygous mutation comfirmed the defects detected by QFM-PCR. CONCLUSION: The approach is a rapid, cost-effective initial method for screening patient samples. It has been used to identify approximately 50% positives of tested samples. The mutations shown were not only small deletion and insertion, but also the single copy exons heterozygous mutation in the RB1 gene which the previously used methods were unable to detect. These features make the technique an attractive approach to clinical diagnosis of gene defects.

Fluorescence↗

PubMed1997

Mutational analysis of the p16 gene in human neuroblastomas.

Neuroblastoma is one of the most frequent tumors in infancy. We analyzed 26 neuroblastomas, two ganglioneuromas, and a neuroblastoma metastasis for mutations and homozygous deletions of the p16 (or MTS1 or CDKN2) gene by means of the polymerase chain reaction (PCR) in combination with the single-strand conformation polymorphism (SSCP) technique and by multiplex PCR analysis. We detected mobility shifts in the SSCP gels in seven cases in the 3 half of exon 2 (named exon 2C) of the p16 gene. By PCR amplification of this particular region and SacII restriction enzyme digestion, we confirmed that those cases had a known polymorphism at codon 140 of the p16 gene. Neither mutations nor homozygous deletions were detected. Our results confirm those of Beltinger et al. (Cancer Res 55:2053-2055, 1995), which showed no p16 mutations or homozygous deletions in 18 primary neuroblastomas and nine tumor-derived cell lines. We conclude that the common pattern of p16 inactivation by homozygous deletion or mutation does not seem to be relevant to the development of neuroblastomas.

Carrier Proteins↗

PubMed2003

Epigenetic detection of human chromosome 14 uniparental disomy.

The recent demonstration of genomic imprinting of DLK1 and MEG3 on human chromosome 14q32 indicates that these genes might contribute to the discordant phenotypes associated with uniparental disomy (UPD) of chromosome 14. Regulation of imprinted expression of DLK1 and MEG3 involves a differentially methylated region (DMR) that encompasses the MEG3 promoter. We exploited the normal differential methylation of the DLK1/MEG3 region to develop a rapid diagnostic PCR assay based upon an individual's epigenetic profile. We used methylation-specific multiplex PCR in a retrospective analysis to amplify divergent lengths of the methylated and unmethylated MEG3 DMR in a single reaction and accurately identified normal, maternal UPD14, and paternal UPD14 in bisulfite converted DNA samples. This approach, which is based solely on differential epigenetic profiles, may be generally applicable for rapidly and economically screening for other imprinting defects associated with uniparental disomy, determining loss of heterozygosity of imprinted tumor suppressor genes, and identifying gene-specific hypermethylation events associated with neoplastic progression.

Chromosomes, Human, Pair 14↗

PubMed2003

Identification of CYP2D6 alleles by single nucleotide polymorphism analysis using pyrosequencing.

OBJECTIVE: To develop a single nucleotide polymorphism (SNP) analysis for identification of cytochrome P450 (CYP)2D6 alleles by pyrosequencing. METHODS: Swedish blood donors ( n=282) were typed for a partial CYP2D6 genotype comprising the alleles *1 (wild type), *2 (2850C>T), *3 (2549A>del), *4 (1846G>A) and *6 (1707T>del) using polymerase chain reaction (PCR) and pyrosequencing analysis. CYP2D6*5 (CYP2D6 deleted) was identified using an established long multiplex PCR method. Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzymatic reactions yields detectable light, which is proportional to the incorporated nucleotides. One feature of typing SNPs by pyrosequencing is that each allelic variant will give a unique sequence. These variants can be readily distinguished by pattern recognition software. RESULTS: Of 281 individuals analysed, 24 (8.5%) were found to be poor metabolisers with two non-functional alleles. This is in the range of 7-10%, previously reported for Caucasians. A total of 126 individuals (45%) had one functional and one non-functional allele and 131 individuals (47%) had two functional alleles. CONCLUSION: Pyrosequencing was found to be a fast and efficient tool for genotyping. The method is robust, reliable, rapid and has high throughput.

Alleles↗

PubMed2005

Sex identification of normal persons and sex reverse cases from bloodstains using FISH and PCR.

UNLABELLED: Sex identification of dry blood is of crucial importance in forensic medicine. SUBJECTS AND METHODS: Sixty normal (with matching phenotypic and genotypic sex) persons (36 males and 24 females), and 7 cases of sex reverse, i.e., persons with one genotypic sex and ambiguous or external genitalia of the opposite sex (3 phenotypic females with Swyer syndrome and the 46,XY karyotype, and 4 phenotypic Klinefelter-like males with the 46,XX karyotype) were subjected to sex identification by FISH and PCR using bloodstains. RESULTS: The FISH technique using an X/Y cocktail probe (DXZI & DYZI, Oncor) has identified the sex correctly in 91.69% of interphase nuclei of the 36 males of the study, and in 92.29% of cells of the 24 females and incorrectly identified the 3 phenotypic females with Swyer syndrome as males and the 4 Klinefelter-like males as females. The 60 normal individuals in the study were correctly typed to their phenotypic sex by the 2 PCR methods used, i.e., the single PCR using the amelogenin sequence specific for the X and Y chromosomes and the multiplex PCR using SRY gene (male-specific) and the AR gene (X-specific). Out of the 7 sex reverse cases, one Klinefelter-like male was incorrectly identified by PCR as female due to the absence of amplification of the SRY gene and the amelogenin male-specific 788 bp fragment. CONCLUSION: The present study demonstrates that both FISH and PCR techniques are fast, easy to perform, reliable and efficient for sex identification but PCR is more accurate. It also emphasises that the sex identified is the genotypic sex which does not necessarily correspond to the phenotypic one and if evidences at the scene of crime indicate opposite sex of the accused, persons with sex reverse have to be ruled out using different X- and Y-specific probes and PCR.

Blood Stains↗

PubMed2006

Evaluation of high-risk human papillomavirus types PCR detection in paired urine and cervical samples of women with abnormal cytology.

BACKGROUND: During the last decade, increasing efforts have focused on HPV detection in self-obtained samples, to increase the overall proportion of patients participating in cervical cancer screening procedures. OBJECTIVES: A clinical evaluation study of an optimized protocol for PCR detection of high-risk human papillomavirus (HPV) types in urine compared with cervical samples in consecutive women referred to the colposcopy clinic with abnormal cervical cytology. STUDY DESIGN: Paired urine and cervical specimens were collected from 100 consecutive women referred to the colposcopy clinic with abnormal cervical cytology and normal urine parameters. In-house and a commercial PCR method for the detection of HPV types 16 and 18, and a commercial multiplex PCR for HPV types 6, 11, 16, 18, and 33 were performed. All HPV cervix-positive/urine-negative paired urine samples were spiked with serial dilutions of cell lines infected with HPV 16 or 18 to test the sensitivity of HPV detection in these urine samples. RESULTS: In all but two cases HPV type 16 was detected. In cancer cases, the urine/cervix HPV detection sensitivity was 88.8%; in cases with high-grade lesions it was 76.5%; and in cases with low-grade lesions it was 45.5%. In all concordant cases the same HPV type was detected in both samples. The urine/cervix HPV detection sensitivity was higher when urine samples contained two or more epithelial cells per field in urine microscopy. HPV detection in 9 cervix-positive but urine-negative urine samples spiked with serial dilutions of HPV-positive cell lines showed that in these cases urine PCR inhibitors did not affect PCR amplification. CONCLUSIONS: A higher urine/cervix HPV detection sensitivity in cancer and high-grade lesions suggests that urine testing could be used to detect HPV mainly when these lesions are present.

Cell Line↗

PubMed2002

Bordetella pertussis PCR: simultaneous targeting of signature sequences.

The absence of analytical controls for polymerase chain reaction (PCR)-based diagnostic tests for Bordetella pertussis limits their clinical utility. In this study, multiplex PCR simultaneously targeted two specific Bordetella pertussis sequences, the chromosomal repeated insertion sequence IS481 (IS) and the pertussis toxin promoter region (PT). A multi-target hybridization-EIA (Hyb-EIA) method in a 96-well microtiter-plate format was used to detect amplicons. Forty-seven (15%) of the 318 nasopharygeal specimens tested positive for at least one DNA target of B. pertussis by PCR, including the 10 known positive samples by culture and/or direct fluorescent antibody (DFA). Forty-six of the 47 PCR positive samples were considered positive for B. pertussis using the consensus interpretation criteria. Simultaneous detection of multiple chromosomal regions may identify false-positive and -negative results due to analytical variations or potential sequence polymorphism, and uncover a wider range of pathogenic strains.

Adolescent↗

PubMed2003

Autonomous detection of aerosolized Bacillus anthracis and Yersinia pestis.

We have developed and tested a fully autonomous pathogen detection system (APDS) capable of continuously monitoring the environment for airborne biological threat agents. The system is designed to provide early warning to civilians in the event of a terrorist attack. The final APDS will be completely automated, offering aerosol sampling, in-line sample preparation fluidics, multiplexed detection and identification immunoassays, and orthogonal, multiplexed PCR (nucleic acid) amplification and detection. The system performance (current capabilities include aerosol collection, multiplexed immunoassays, sample archiving, data reporting, and alarming) was evaluated in a field test conducted in a Biosafety Level 3 facility, where the system was challenged with, and detected, a series of aerosolized releases containing two live, virulent biological threat agents (Bacillus anthracis and Yersinia pestis). Results presented here represent the first autonomous, simultaneous measurement of these agents.

Aerosols↗

PubMed2003

Genotype and allele frequencies of TPMT, NAT2, GST, SULT1A1 and MDR-1 in the Egyptian population.

AIMS: The goal of this study was to determine the frequencies of important allelic variants in the TPMT, NAT2, GST, SULT1A1 and MDR-1 genes in the Egyptian population and compare them with the frequencies in other ethnic populations. METHODS: Genotyping was carried out in a total of 200 unrelated Egyptian subjects. TPMT*2 was detected using an allele-specific polymerase chain reaction (PCR) assay. TPMT*3C and NAT2 variants (*5,*6 and *7) were detected using an allele-specific real-time PCR assay. Detection of GSTM1 and GSTT1 null alleles was performed simultaneously using a multiplex PCR assay. Finally, a PCR-restriction fragment length polymorphism assay was applied for the determination of TPMT*3A (*3B), SULT1A1*2 and MDR-1 (3435T) variants. RESULTS: Genotyping of TPMT revealed frequencies of 0.003 and 0.013 for TPMT*3A and TPMT*3C, respectively. No TPMT*2 or *3B was detected in the analysed samples. The frequencies of specific NAT2 alleles were 0.215, 0.497, 0.260 and 0.028 for *4 (wild-type), *5 (341C), *6 (590A) and *7 (857A), respectively. GSTM1 and GSTT1 null alleles were detected in 55.5% and 29.5% of the subjects, respectively. SULT1A1*2 was detected at a frequency of 0.135. Finally, the frequencies of the wild-type allele (3435C) and the 3435T variant in the MDR-1 gene were found to be 0.6 and 0.4, respectively. CONCLUSIONS: We found that Egyptians resemble other Caucasians with regard to allelic frequencies of the tested variants of NAT2, GST and MDR-1. By contrast, this Egyptian population more closely resemble Africans with respect to the TPMT*3C allele, and shows a distinctly different frequency with regard to the SULT1A1*2 variant. The predominance of the slow acetylator genotype in the present study (60.50%) could not confirm a previously reported higher frequency of the slow acetylator phenotype in Egyptians (92.00%), indicating the possibility of the presence of other mutations not detectable as T341C, G590A and G857A. The purpose of our future studies is to investigate for new polymorphisms, which could be relatively unique to the Egyptian population.

Arylamine N-Acetyltransferase↗