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PubMed1998

Multiplex PCR for the detection of Mycoplasma fermentans, M. hominis and M. penetrans in cell cultures and blood samples of patients with chronic fatigue syndrome.

A multiplex polymerase chain reaction (PCR) was initially developed to detect the presence of mycoplasma genus DNA sequences in cell cultures and to differentiate between three human pathogenic mycoplasma species simultaneously. The assay in turn, proved to be a useful tool for the detection of mycoplasma infection in human DNA samples. One set of oligonucleotide primers which are specific for a highly conserved region among all members of the genus mycoplasma along with three other primer sets which are specific for Mycoplasma fermentans, Mycoplasma hominis andMycoplasma penetrans species were used in this assay. The sensitivity of detection was determined by infecting peripheral blood mononuclear cells (PBMC) of healthy individuals with known bacterial copy numbers from each species, extracting the DNA, and subjecting 1 microgram of DNA from each sample to 40 cycles of amplification. By using agarose gel electrophoresis the detection level was determined to be 7, 7, 9 and 15 mycoplasma cells per microgram of human genomic DNA for M. genus,M. fermentans, M. hominis and M. penetrans, respectively. The assay was applied to DNA extracted from the PBMCs of individuals suffering from chronic fatigue syndrome (CFS) (n=100) as determined by the Center for Disease Control (CDC) criteria, and compared to healthy individuals (n=100). The percentage of M. genus infection was found to be 52% in CFS patients and only 15% in healthy individuals. Mycoplasma fermentans, M. hominis andM. penetrans were detected in 32, 9 and 6% of the CFS patients while they were detected in 8, 3 and 2% of the healthy control subjects, respectively. This assay provides a rapid and cost efficient procedure to screen either cell cultures or clinical samples for the presence of three potentially pathogenic species of mycoplasma with a high level of sensitivity and specificity.

Blood↗
PubMed2000

Multiplex PCR detection of Campylobacter jejuni and Arcobacter butzleri in food products.

Arcobacter is a recently described species, previously considered part of the Campylobacter family. A sensitive assay such as that provided by PCR could help to distinguish the closely related Arcobacter from Campylobacter. A PCR method to specifically detect both Campylobacter jejuni and Arcobacter butzleri in the same reaction tube has been developed. C. jejuni and A. butzleri were inoculated into a range of dairy products, raw and ready-to-eat foods. The presence of these two organisms was detected in these test foods by the multiplex PCR assay. A product of 159 bp was apparent when C. jejuni was present, while a 1223 bp product was seen when A. butzleri was present. When both organisms were present, both bands could be detected on the agarose gel. All organisms were confirmed by standard microbiological methods. There was complete agreement between the PCR and standard methods. This PCR assay will allow detection of both organisms within the same PCR tube and can be performed within an 8 h day. The presence of these two human pathogens, which are difficult to distinguish by standard biochemical methods, can now be identified using this PCR assay.

Animals↗
PubMed2001

A multiplex-PCR for the diagnosis of contagious agalactia of sheep and goats.

A multiplex polymerase chain reaction (m-PCR) assay was optimized for the simultaneous detection of several species of small ruminant mycoplasmas. Two sets of oligonucleotide primers specific for Mycoplasma agalactiae (Ma) and Mycoplasma "mycoides" cluster (M.m. cluster) were used in the test. The m-PCR was able to amplify a 375-bp fragment of Ma chromosomal DNA and a 257-260-bp fragment of M.m. cluster chromosomal DNA. Four reference strains (M. agalactiae, M. mycoides subsp. mycoides LC, M. capricolum subsp. capricolum, M. mycoides subsp. capri) and 56 samples (44 milk samples, two nasal swabs, six ocular swabs, three vaginal swabs and one sample of fibrinous exudate from carpal joint), from sheep and goats with clinical signs of contagious agalactia (CA), were examined. The m-PCR confirmed the identification of reference strains and allowed to identify, of the 43 positive samples examined, 35 Ma strains, 12 M. "mycoides" cluster strains; in four samples both Ma and mycoplasmas of M. "mycoides" cluster were revealed. The m-PCR was able to detect 1 pg of mycoplasma DNA. The specificity and sensitivity of the m-PCR suggest its use for the "routine" diagnosis of CA.

Animals↗
PubMed2001

Development of a multiplex PCR for the detection of Haplosporidium nelsoni, Haplosporidium costale and Perkinsus marinus in the eastern oyster (Crassostrea virginica, Gmelin, 1971).

Haplosporidium costale (SSO), Haplosporidium nelsoni (MSX) and Perkinsus marinus (Dermo) have caused oyster mortality on the North American east coast since the 1950s. Currently, the monitoring of oyster populations for these pathogens depends on histopathology for H. nelsoni, H. costale and the Ray/Mackin assay for P. marinus. In this study we describe the development and optimization of a multiplex polymerase chain reaction (MPCR) for the detection of H. nelsoni, H. costale and P. marinus. In addition, we determine its specificity and sensitivity. The MPCR clearly detects and differentiates the protozoan pathogens. There was no cross-reactivity between these species. The MPCR was able to detect DNA from H. nelsoni as low as 10 fg and P. marinus and H. costale as low as 1 pg. The MPCR allows for the detection of H. nelsoni, H. costale and P. marinus in a single PCR reaction.

Animals↗
PubMed2002

Serogroup specific single and multiplex PCR with pre-enrichment culture and immuno-magnetic bead capture for identifying strains of D. nodosus in sheep with footrot prior to vaccination.

The identification of Dichelobacter nodosus present in a flock is a prerequisite to specific (autogenous) vaccination. Current methods of identification of the serogroup present in a population requires that the organisms be isolated, identified visually in mixed culture on streak plates, subcultured to purify and subjected to antigenic analysis. This process takes at least 3 to 4 weeks. This study describes the development of a simple and rapid serogroup specific PCR test for D. nodosus. A common forward primer was designed from the conserved amino-terminal region of the fimbrial gene (fimA) and 9 (A-I) serogroup specific reverse primers were designed from the carboxy-terminal regions of fimA of the different serogroups. To verify the specificity within D. nodosus, each specific primer pair was tested in PCR against 18 serogroups/serotypes (prototypes) and found to be specific for all the serotypes within the homologous serogroups. Eighty four other bacterial strains, either commonly occurring in sheep or found in the environment of sheep, and including organisms related taxonomically to D. nodosus, were used to check the specificity of these assays. They were found to be specific for D. nodosus as none of the 84 bacterial stains reacted. These primers detected 1 pg of purified chromosomal DNA, or 50-100 cells of D. nodosus in crude lysates. Sensitivity was markedly improved when an immuno-magnetic capture was employed. Single tube multiplex PCRs were tested with different combinations of common forward primer and groups of 3, 4 or 5 reverse primers chosen so that amplicon size for each reaction product was different. These were able to amplify DNA of isolates from all the relevant serogroups included in the reactions. These tests were evaluated with samples taken directly from lesions of footrot, either directly or preceded by DNA purification, immuno-magnetic capture, enrichment broth culture and culture on hoof agar media. Of these methods only PCR on mixed colonies from 4-day-old cultures on 4% hoof agar media yielded results of practical value.

Animals↗
PubMed2002

Application of a convenient DNA extraction method and multiplex PCR for the direct detection of Staphylococcus aureus and Yersinia enterocolitica in milk samples.

The application of PCR for the direct and sensitive detection of food-borne pathogens is largely affected by the quality of the template DNA prepared from food samples. In the present study, a chemical extraction method of bacterial DNA from spiked milk samples for the direct detection of Staphylococcus aureus and Yersinia enterocolitica was evaluated by PCR. Gene specific primers were designed to target the nuclease (nuc) and the attachment invasion locus (ail) genes of S. aureus and Y. enterocolitica, respectively and used in PCR. A combination of organic solvents, detergents and alkali in the DNA extraction method permitted a detection limit of 10 cfu ml(-1) milk for both the pathogens. When equal numbers of S. aureus and Y. enterocolitica were spiked in milk samples, the individual detection limit was determined to be 10(3) cfu ml(-1) milk. Simultaneous amplification of 482 and 359 bp fragments of the nuc and ail genes was obtained using the primer pairs in a single reaction. Multiplex PCR enabled the detection of 10(4) cfu ml(-1) milk of S. aureus and Y. enterocolitica without any pre-enrichment step. A combination of conventional isolation technique and PCR using DNA extracted by the proposed method was used to test raw milk samples for possible contamination with S. aureus and Y. enterocolitica. The presence of S. aureus in the tested samples was indicated by both the methods while Y. enterocolitica could not be detected in any of the samples. The template DNA extraction method developed in this study is rapid, sensitive and avoids interference from potential PCR inhibitors and demonstrates the potential of detecting multiple pathogens in milk samples without any enrichment.

Animals↗
PubMed2025

Multiplexed Dual-Color Fluorescence-Based Distinction Between Nuclear Trapping and Translocation of FOXO3.

FOXO3 is a transcription factor that mainly exerts its functions in the cell nucleus. The amino acid sequence of FOXO3 contains a nuclear localization sequence (NLS) and a nuclear export sequence (NES) allowing for nuclear/cytoplasmic shuttling that plays an important role in regulating FOXO3 activity. Nuclear accumulation of FOXO3 proteins can be the result of translocation to the nucleus triggered by upstream regulatory input or trapping of FOXO3 within the nucleus through the inhibition of its nuclear export via the receptor CRM1. In order to distinguish these two modes of FOXO3 activation, we have generated a multiplexed assay. The development of this platform includes a reporter cell line that monitors CRM1 activity by using RFP-labeled HIV-1 Rev. protein with a strong heterologous NES. Simultaneously, the intracellular localization of FOXO3 can be monitored by a second cell line stably expressing GFP-FOXO3. Here we describe a detailed protocol on how to co-culture these reporter cell lines and use them to interrogate compound-induced FOXO3 activation in order to understand the mode of action.

Forkhead Box Protein O3↗
PubMed1994

Multiplex PCR screening detects small p53 deletions and insertions in human ovarian cancer cell lines.

Mutations at the p53 tumor suppressor gene locus are a frequent genetic alteration associated with human ovarian carcinoma. Little information exists regarding whether mutational events occur other than point mutations and large deletions, causing loss of heterozygosity. Small intragenic deletions and insertions in the p53 gene have been observed in various human neoplasias. We developed a multiplex polymerase chain reaction (MPCR) screening assay to amplify the complete p53 coding region from genomic DNA in a single step. Deletions and/or insertions were found in six out of 11 newly established ovarian carcinoma cell lines. MPCR detected deletions as small as 2 bp, as confirmed by nucleotide sequence analysis. Most of the observed alterations (6/7) were homozygous or hemizygous. Structural aberrations of the p53 gene possibly leading to loss of p53 cell cycle control may be a consequence of a slipped-mispairing mechanism in rapid DNA replication during repetitious ovulation and wound repair of ovarian epithelial cells. MPCR may be a valuable tool for screening for possible p53 deletion and insertion mutations not only in ovarian cancer but also in other malignancies.

Base Sequence↗
PubMed1992

Detection of small RB1 gene deletions in retinoblastoma by multiplex PCR and high-resolution gel electrophoresis.

Loss of function of both copies of the RB1 gene is a causal event in the development of retinoblastoma. The predisposition to this tumor can be inherited as an autosomal dominant trait. Direct detection of the genetic defect is important for presymptomatic DNA diagnosis and genetic counseling in families with hereditary retinoblastoma. We have used multiplex polymerase chain reaction and high-resolution polyacrylamide gel electrophoresis to detect RB1 gene deletions as small as one base pair. By using three independent sets of amplification reactions, which cover 26% of the RB1 gene coding region, we identified RB1 gene deletions in the DNA of peripheral blood cells in 3 out of 24 (12.5%) unrelated patients with hereditary retinoblastoma. In one case, formalin-fixed paraffin-embedded tumor material was also used to detect the mutation. Sequencing of the mutated alleles revealed deletions of 1, 3 and 10 base pairs. Each deleted region was flanked by direct repeats.

Amino Acid Sequence↗
PubMed1990

Screening for steroid sulfatase (STS) gene deletions by multiplex DNA amplification.

Deletions are the most common molecular defect in steroid sulfatase (STS) deficiency. We describe the application of multiplex DNA amplification, by polymerase chain reaction, for deletion screening in patients with STS deficiency (STS-PCR). Genomic DNA from 38 unrelated patients was amplified using two sets of primers, corresponding to the 5' and the 3' ends of the STS gene. The analysis of the amplified products was always consistent with the results obtained by Southern analysis. This method represents a sensitive fast non-radioactive test for detecting STS gene deletions.

Arylsulfatases↗
PubMed1990

Hypoplasia of posterior spinal roots and dorsal spinal tracts with arthrogryposis multiplex congenita.

We report a male infant with arthrogryposis multiplex congenita (AMC) who survived for 19 weeks following birth at 36 weeks gestational age. No heritable or acquired cause of neuromuscular disease was found. He manifested joint contractures of upper and lower extremities, diffuse hypotonia requiring ventilatory support, and areflexia; the general examination also showed facial dysmorphisms, and an ichthyotic rash. Pathological examination of the brain and spinal cord revealed severe hypoplasia of dorsal roots and posterior columns, nondecussation of pyramidal tracts, and anterior horns of an unusual configuration; the brain was normal, and the cerebellum contained Purkinje cell heterotopias. Muscle spindles could not be identified. To our knowledge, these spinal cord abnormalities in association with AMC have not been previously reported, thus raising interesting speculations about the possible role of such abnormalities in the pathogenesis of AMC.

Arthrogryposis↗
PubMed1988

Decreased number of alpha-motoneurons in the cervical intumescence of calves with arthrogryposis multiplex congenita of both thoracic limbs.

The number of alpha-motoneurons in the spinal cord segments C6 to Th2 of five calves with Arthrogryposis Multiplex Congenita (AMC) and of five normal calves was compared. A statistically highly significant reduction in the number of alpha-motoneurons in segment C6 was observed in the affected calves. Two hypotheses to explain the effect of these findings were proposed. Firstly, it was thought that AMC may result from paresis of some muscles of the girdle, shoulder and elbow. Secondly, it was postulated that the decrease in the number of alpha-motoneurons in C6 may have induced a radial nerve dysfunction resulting in AMC.

Animals↗
PubMed1985

Congenital muscular dystrophy associated with lethal arthrogryposis multiplex congenita.

Two unrelated patients with severe arthrogryposis multiplex congenita (AMC) who died perinatally, are presented. In both, postmortem examination revealed an intact nervous system and striking dystrophic muscle changes, consistent with congenital muscular dystrophy (CMD). Few similar cases have been reported before, but since the condition is not well known, it seems probable that in the past many have been labeled as mere multiple malformations. The possibility of an underlying muscular disorder, either primary myopathic or neurogenic should be considered in any patient with early lethal AMC. Our findings confirm that the fetal akinesia-arthrogryposis sequence is a nonspecific clinical syndrome resulting from various causes of muscular inactivity in utero. The main objective of this report is to provide reasonable guidelines on how to approach the problem of classification. We favor a pathogenetic approach, depending upon careful sampling of the central nervous system and skeletal muscles at autopsy.

Abnormalities, Multiple↗
PubMed1994

Evaluation of an automated DNA profiling system employing multiplex amplification of four tetrameric STR loci.

We have examined the performance and reproducibility of an automated DNA profiling system which is based on the multiplex amplification of 4 tetrameric STR loci-HUMVWFA31/A. HUMTH01, HUMF13A1 and HUMFES/FPS. The system was able to type 100 pg of purified, undegraded, genomic DNA. At lower concentrations of DNA (below 100 pg), allelec drop-out occurred due to stochastic differences in allele copy number. Minor variation of individual PCR reagent concentrations or cycling temperatures did not result in a significant effect on the efficiency of amplification of any of the 4 loci in the quadruplex system. More substantial variation of reagent concentrations or cycling temperatures outside the optimum range of the system resulted in a reduction or complete loss of signal for one or more loci. This was also observed at high ionic strength or extreme pH. However, under all reagent concentrations and conditions studied, no artefact bands that could potentially result in the mistyping of a sample were apparent within the read region (130-240 bases) of the gel. Evaluation of both native and denaturing polyacrylamide gels revealed that, although native gels displayed faster run times, the sizing precision of such gels for certain STR loci was lower than that of denaturing gels. Also, artefact bands may be present within the read region of native gels. In conclusion the quadruplex amplification system described, coupled with automated fluorescence-based detection on denaturing polyacrylamide gels, appeared to be a robust and reliable system for individual identification.

Alleles↗
PubMed1996

The validation of a 7-locus multiplex STR test for use in forensic casework. (I). Mixtures, ageing, degradation and species studies.

We have evaluated a multiplex STR system for routine forensic use, which co-amplifies six short tandem repeat (STR) loci; HUMTH01, D21S11, D18S51, D8S1179, HUMVWF31/A and HUMFIBRA (FGA), in conjunction with the X-Y homologous gene Amelogenin. Analysis of PCR products employs denaturing polyacrylamide gels coupled with fluorescent labelled primers and detection is undertaken on ABD 373A automated sequencers. The technique was shown to be robust and reproducible when samples were analysed under conditions consistent with those encountered in a forensic environment. The system was demonstrated to be human specific and is suitable for use with both aged and degraded material. Somatic stability was proven with a wide range of tissue types and we were able to detect mixtures at ratios between 1:10 and 10:1. During this study no incidence of sample mis-typing due to allelic or locus drop-out was observed. Furthermore, although additional artefact bands were occasionally encountered these did not interfere with the interpretation of results. The performance of the system with poor quality samples demonstrated its suitability as a powerful tool in forensic investigation.

Adult↗
PubMed1994

Sequential multiplex amplification (SMA) of genetic loci: a method for recovering template DNA for subsequent analyses of additional loci.

A method called Sequential Multiplex Amplification (SMA) has been developed whereby a limited amount of DNA extracted from a sample can be reutilized for several single polymerase chain reaction (PCR) amplifications. The method involves recovery of genomic template DNA by microfiltration of PCR-amplified samples. Up to 5 different loci have been typed, each in a single system PCR-based assay, beginning with a test quantity of 5 ng template DNA. Genotypes of the DNA donors were compared with those obtained from individual amplifications and shown to be identical. This could be a useful technique for typing a number of loci from a limited amount of DNa and to recover template DNA from samples previously subjected to PCR. Obviously, when small quantities of template DNA are available, this technique can prove quite useful.

Base Sequence↗
PubMed1995

Swiss population data and forensic efficiency values on 3 tetrameric short tandem repeat loci-HUMTH01, TPOX, and CSF1PO-derived using a STR multiplex system.

Allele and genotype frequencies for 3 tetrameric short tandem repeat loci were determined in a Swiss population sample (n = 100) using the GenePrint STR Multiplex System, electrophoresis of the PCR products in DNA sequencing gels and subsequent detection of allelic fragments by silver staining. The loci are HUMTH01, TPOX, and CSF1PO. The observed heterozygosities are 83.0%, 60.0%, and 72.0%, respectively. The discrimination power determined for the individual loci is 0.914, 0.780, and 0.860, respectively, and the combined discrimination power for the triplex is 0.997. All loci meet Hardy-Weinberg expectations and after Bonferroni correction there was no evidence that the population sample deviates from expectations of independence. Moreover, independence of alleles at these STR loci with other PCR-based loci derived from the same Swiss population sample, previously reported, were considered. These loci were DQA1, LDLR, GYPA, HBGG, D7S8, GC and D1S80. Again, after Bonferroni correction there was no evidence that the population sample deviates from expectations of independence among alleles at the 10 different PCR-based loci. Thus, the allelic frequency data can be used in human identity testing to estimate the frequency of a multiple PCR-based DNA profile in the Swiss population.

Alleles↗