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PubMed1993

Deletion detection for diagnosis of Duchenne muscular dystrophy in the Japanese population--comparison between the polymerase chain reaction and the Southern blot analysis.

We compared the efficacy of the multiplex PCR with that of the cDNA analysis for detection of deletions of the DMD gene in the Japanese patients. Thirty males with DMD from 27 Japanese families were studied by the multiplex PCR, and 24 of them were also investigated by Southern blot analysis. We used five dystrophin cDNA probes for deletion analysis. A total of 19 regions were amplified by the PCR to detect deletions, 9 regions by the method of Chamberlain et al. and another 10 regions by the method of Beggs et al. Deletions were detected in 14 (52%) out of 27 DMD families by the PCR. Southern blot analysis detected deletions in 14 (64%) out of 22 families. Thirteen (93%) of the 14 DMD families with deletions detected by Southern blotting were also confirmed by the multiplex PCR. Provided care is taken in cases where the deletion is limited to a single exon, the multiplex PCR appears to be an efficient and useful alternative to conventional Southern blot analysis for detecting deletions during the prenatal and postnatal diagnosis of DMD.

Asian People↗

PubMed1997

PCR amplification of the mitochondrial DNA minisatellite region to detect Schistosoma mansoni infection in Biomphalaria glabrata snails.

Schistosoma infection in Biomphalaria glabrata can be detected by either exposing snails to light to induce cercarial shedding or by squeezing them between glass slides to detect parasites in the digestive gland and other regions. The methods available are inefficient for identification of prepatent infections and do not allow the diagnosis of infection in snails that die before arriving in the laboratory. Furthermore, infection is undetectable after migration of sporocysts from the head-foot region of the snail. We examined the use of polymerase chain reaction (PCR) amplification of minisatellite repeats from Schistosoma mansoni mitochondrial DNA (mtDNA) to identify snail infection. We found that amplification of mtDNA under low stringency conditions (LS-PCR) allowed for the identification of specific S. mansoni infection in Biomphalaria snails. To confirm these results, specific amplification reactions were performed using 2 sets of primers that allowed for the diagnosis of infection and an internal control of the reaction (multiplex PCR). Results obtained using multiplex PCR demonstrated the ability of the assay to detect S. mansoni-specific infection. Thus, LS-PCR as well as specific multiplex PCR allow for the detection of prepatent infections and show high specificity for S. mansoni in comparison with other trematode infections in either living or dead snails.

Animals↗

PubMed2006

Detecting exon deletions and duplications of the DMD gene using Multiplex Ligation-dependent Probe Amplification (MLPA).

OBJECTIVES: To evaluate the efficacy of Multiplex Ligation-dependent Probe Amplification (MLPA) technique in comparison with the traditional multiplex PCR assay in detection of exon deletions and duplications of the DMD gene. DESIGN AND METHODS: The sensitivity and accuracy of MLPA were assessed and compared with the multiplex PCR in a total of 63 subjects including 43 subjects with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) and 20 female carriers. RESULTS: MLPA was able to detect all the known deletions and duplications; it detected four additional mutations that had been missed by multiplex PCR. In addition, the extent of the deletions and duplications could be more accurately defined which in turn facilitated a genotype-phenotype correlation. CONCLUSIONS: MLPA is superior to multiplex PCR. It should be the method of choice for the detection of exon deletions and duplications of the DMD gene in patients with DMD or BMD, as well as in female carriers.

Dystrophin↗

PubMed2001

Genetic characterisation of invasive breast cancer: a comparison of CGH and PCR based multiplex microsatellite analysis.

AIMS: Comparative genomic hybridisation (CGH) is a reliable tool to gain an overview of all unbalanced chromosomal alterations within a tumour. Nevertheless, the high numbers of tumour cells required and the comparatively low resolution are drawbacks of this technique. Polymerase chain reaction (PCR) based multiplex microsatellite analysis represents a semi-automated, highly reproducible method, which requires small amounts of tumour cells. This is a comparative study of CGH and microsatellite analysis. METHODS: Eighty one samples of invasive breast cancer were investigated by two sensitive multiplex PCRs containing three microsatellites each of six markers (D6S261, D11S907, D6S300, D11S927, D8S272, and D11S925), and two additional microsatellite markers located within intron 1 of the epidermal growth factor receptor gene (egfr) and p53 (p53CA). RESULTS: At least one example of loss of heterozygosity was detectable in all breast cancer tissues. However, the overall rate of accordance between the two methods tested was only 61%. An increasing rate of the number of genetic alterations in each case was mirrored by a constantly increasing fractional allelic loss index. CONCLUSIONS: PCR based multiplex microsatellite analysis using this panel of eight microsatellite markers not only enables the characterisation of cells that have malignant potential in a high frequency of patients with breast cancer, but can also give an estimate of the degree of genetic progression.

Breast Neoplasms↗

PubMed2005

Qualitative and quantitative PCR methods for event-specific detection of genetically modified cotton Mon1445 and Mon531.

Based on the DNA sequences of the junctions between recombinant and cotton genomic DNA of the two genetically modified (GM) cotton varieties, herbicide-tolerance Mon1445 and insect-resistant Mon531, event-specific primers and probes for qualitative and quantitative PCR detection for both GM cotton varieties were designed, and corresponding detection methods were developed. In qualitative PCR detection, the simplex and multiplex PCR detection systems were established and employed to identify Mon1445 and Mon531 from other GM cottons and crops. The limits of detection (LODs) of the simplex PCR were 0.05% for both Mon1445 and Mon531 using 100 ng DNA templates in one reaction, and the LOD of multiplex PCR analysis was 0.1%. For further quantitative detection using TaqMan real-time PCR systems for Mon1445 and Mon531, one plasmid pMD-ECS, used as reference molecule was constructed, which contained the quantitative amplified fragments of Mon1445, Mon531, and cotton endogenous reference gene. The limits of quantification (LOQs) of Mon1445 and Mon531 event-specific PCR systems using plasmid pMD-ECS as reference molecule were 10 copies, and the quantification range was from 0.03 to 100% in 100 ng of the DNA template for one reaction. Thereafter, five mixed cotton samples containing 0, 0.5, 0.9, 3 and 5% Mon1445 or Mon531 were quantified using established real-time PCR systems to evaluate the accuracy and precision of the developed real-time PCR detection systems. The accuracy expressed as bias varied from 1.33 to 8.89% for tested Mon1445 cotton samples, and from 2.67 to 6.80% for Mon531. The precision expressed as relative standard deviations (RSD) were different from 1.13 to 30.00% for Mon1445 cotton, and from 1.27 to 24.68% for Mon531. The range of RSD was similar to other laboratory results (25%). Concluded from above results, we believed that the established event-specific qualitative and quantitative PCR systems for Mon1445 and Mon531 in this study are acceptable and suitable for GM cotton identification and quantification.

Animals↗

PubMed2001

Evaluation of two PCR-based techniques for molecular epidemiology in Finland, a high-endemic area with four sympatric Trichinella species.

Trichinella larvae collected from wildlife, domestic and synanthropic animals in Finland were identified to species by two molecular techniques: Random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) and the recently described multiplex PCR. The RAPD-PCR was very sensitive to the sub-optimal preservation muscle larvae and resulting in weak and smeared bands on the gels for such material. However, the same samples yielded easily recognizable bands in the multiplex PCR; this latter technique is then recommended for epidemiological studies, especially when the preservation of the samples is sub-optimal. For larvae in good condition the unequivocal bands obtained by multiplex was the easiest identifiable. Four species of Trichinella were identified in the material: T. spiralis, T. nativa, T. britovi, and T. pseudospiralis. Trichinella britovi is a new record for Finland, and T. pseudospiralis is a new record for Northern Europe. Mixed infections between T. britovi and T. spiralis, T. nativa and T. spiralis, and between T. britovi and T. nativa were detected; this is the first record of a mixed infection between T. spiralis and T. nativa in a naturally infected host. Raccoon dogs were the only host species from which all of the four Trichinella species were detected. Trichinella spiralis was found in both domestic animals and wildlife, but none of the sylvatic Trichinella species were detected in domestic pig.

Animals↗

PubMed1997

Use of different PCR-based DNA fingerprinting techniques and pulsed-field gel electrophoresis to investigate the epidemiology of Acinetobacter calcoaceticus-Acinetobacter baumannii complex.

Acinetobacter calcoaceticus-Acinetobacter baumannii complex is an important nosocomial pathogen for which optimal typing methods in epidemiologic investigations have not been defined. We compared DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) with different PCR-based DNA fingerprinting techniques, including enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction (PCR), repetitive extragenic palindromic (REP) PCR, arbitrary-primed PCR with primer M13, and multiplex PCR with primers REP-1, REP-2 and M13, for characterization of 98 clinical isolates (including 10 apparent outbreak-related isolates and 68 presumed epidemiologically unrelated isolates) in a tertiary-care hospital over a 4-year period. The PFGE patterns after Smal restriction of the bacterial DNA were analyzed by computer software (Gelcompar) using the unweighted pair group method with arithmetic averages clustering and the Dice coefficient. A cluster of 48 isolates (cluster A), including 9 outbreak isolates, linked at a level of 83.4% similarity was observed. This epidemic strain and its variants were also found among the 68 presumed epidemiologically unrelated isolates, and this may represent ongoing endemic infection in this institution. The discrimination index for the PCR-based DNA fingerprinting techniques was 0.75 for enterobacterial repetitive intergenic consensus 1, 0.71 for M13, 0.77 for REP-1, 0.77 for REP-2, and 0.87 for multiplex PCR. The discriminatory power of PFGE was found to be higher than those of PCR-based techniques. It was concluded that both PFGE and PCR-based fingerprinting are useful for typing of A. calcoaceticus-A. baumannii complex. However, PFGE can detect minor mutations among outbreak strains, and this is important for epidemiological study of this species in a complex endemic setting.

Acinetobacter↗

PubMed2006

A successful strategy for Preimplantation Genetic Diagnosis of beta-thalassemia and simultaneous detection of Down's syndrome using multiplex fluorescent PCR.

OBJECTIVES: Preimplantation Genetic Diagnosis (PGD) is an alternative to prenatal diagnosis providing couples the chance to start a pregnancy with an unaffected fetus. The objective of the present study was to develop and apply quick, sensitive and accurate single cell PCR protocols for PGD of beta-thalassemia and Down's syndrome detection. MATERIAL AND METHOD: Two couples carrying beta-thalassemia codon41-42 mutation underwent routine IVF procedures. Embryo biopsy was performed on Day-3 post-fertilisation and single cell multiplex fluorescent PCR was employed for mutation analysis, contamination detection and diagnosis of trisomy 21 cases. RESULTS: Seventeen embryos were tested in two clinical PGD cycles. This resulted in the first birth following PGD for a single gene disorder in Thailand and South East Asia, confirmed by prenatal testing. Two embryos were shown to be affected by Down's syndrome. CONCLUSION: Successful strategy for PGD of beta-thalassemia and Down's syndrome detection using multiplex fluorescent PCR was introduced.

Adult↗

PubMed2001

[Rapid detection of the common alpha-thalassemia-2 determinants by PCR assay].

OBJECTIVE: Most frequent molecular lesions of alpha-thalassemia-2 determinants are deletions of one alpha-globin gene. Satisfactory PCR methodologies for detecting the deletions are required for molecular diagnosis and genetic screening since there was no internal control in most published PCR-based strategies. The purpose of this study is to develop a reliable PCR protocol specific for the two common alpha-thalassemia-2 determinants with internal control. METHODS: The multiple repeat elements and the high GC-content of the alpha-globin locus impose severe limitations on designing suitable primers and optimizing stable conditions for PCR. In this study, two multiplex PCR systems were successfully set up. One was designed to detect the rightward deletion (-alpha(3.7)/) with two pairs of primers including one newly optimized pair for amplification of the internal standard to indicate the success of failure of PCR amplification. The other, to the leftward deletion(-alpha(4.2)/) with three primers, which were designed according to the newly sequenced data of the -alpha(4.2) and HbQ-alpha(4.2) deletions in this lab(Genbank Accession No. AF221717). In the PCR system, one is used as a common upstream primer and the other two are used as specific downstream primers for typing the normal allele and the deletion one, respectively. RESULTS: Easily interpretable, unambiguous amplifications were observed by using the multiplex PCR systems for the detection of the two common alpha-thalassemia-2 determinants. The three or four primers were run in the same tube under the same condition and both of these two systems could be used at the same thermal cycle parameters. For typing the rightward deletion, a mutant-specific amplification of 1.7 kb and a 1140 bp amplified band as a normal and system control were produced. For typing the leftward deletion, two PCR-amplified bands, a 956 bp fragment specific for a -alpha(4.2) gene and a 1140 bp one for a normal allele were found. CONCLUSION: Two sets of PCR systems with internal controls for detecting the most common two alpha-thalassemia-2 determinants have been established and may be suitable for molecular diagnosis and population screening.

Base Sequence↗

PubMed2001

Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR.

A single-tube 5' nuclease multiplex PCR assay was developed on the ABI 7700 Sequence Detection System (TaqMan) for the detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae from clinical samples of cerebrospinal fluid (CSF), plasma, serum, and whole blood. Capsular transport (ctrA), capsulation (bexA), and pneumolysin (ply) gene targets specific for N. meningitidis, H. influenzae, and S. pneumoniae, respectively, were selected. Using sequence-specific fluorescent-dye-labeled probes and continuous real-time monitoring, accumulation of amplified product was measured. Sensitivity was assessed using clinical samples (CSF, serum, plasma, and whole blood) from culture-confirmed cases for the three organisms. The respective sensitivities (as percentages) for N. meningitidis, H. influenzae, and S. pneumoniae were 88.4, 100, and 91.8. The primer sets were 100% specific for the selected culture isolates. The ctrA primers amplified meningococcal serogroups A, B, C, 29E, W135, X, Y, and Z; the ply primers amplified pneumococcal serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10A, 11A, 12, 14, 15B, 17F, 18C, 19, 20, 22, 23, 24, 31, and 33; and the bexA primers amplified H. influenzae types b and c. Coamplification of two target genes without a loss of sensitivity was demonstrated. The multiplex assay was then used to test a large number (n = 4,113) of culture-negative samples for the three pathogens. Cases of meningococcal, H. influenzae, and pneumococcal disease that had not previously been confirmed by culture were identified with this assay. The ctrA primer set used in the multiplex PCR was found to be more sensitive (P < 0.0001) than the ctrA primers that had been used for meningococcal PCR testing at that time.

ATP-Binding Cassette Transporters↗

PubMed2006

Multiplex PCR-based reverse line blot hybridization assay to identify 23 Streptococcus pneumoniae polysaccharide vaccine serotypes.

We developed a multiplex PCR-based reverse line blot assay to identify 23 pneumococcal serotypes represented in the polysaccharide vaccine, using 334 well-characterized isolates, representing all 90 serotypes, and 268 "unknowns." The assay identified all target serotypes, but 11, which cross-react with 1 to 4 nonvaccine serotypes, could be distinguished using serotype-specific antisera.

Antibodies, Bacterial↗

PubMed2000

A method for homogeneous color-compensated genotyping of factor V (G1691A) and methylenetetrahydrofolate reductase (C677T) mutations using real-time multiplex fluorescence PCR.

OBJECTIVES: To set up an optimized multiplex polymerase chain reaction for real-time genotyping of the prothrombotic risk factors methylenetetrahydrofolate reductase C677T and factor V Leiden on the LightCycler. DESIGN AND METHODS: Novel primer and probe sets were designed on the basis of thermodynamic double-strand DNA stability calculations. Detection probes were labeled with LC-Red640 or Cy5.5 dye. RESULTS: The polymerase chain reaction efficiency was reduced in multiplex polymerase chain reaction but this could be overcome by the design of novel amplification primers. The selection of detection probes with a lower melting temperature (T(m)) and high Delta T(m) improved the discrimination of heterozygous samples. Color compensation was not compromised by either the use of the Cy5.5 dye or different fluorescein linker chemistries. CONCLUSIONS: Probes with a Delta T(m) of 5 degrees C or more between the matched and mismatched state are desirably for genotyping. Such probes can be selected by using a priori calculations based on the thermodynamic nearest neighbor model.

DNA Mutational Analysis↗

PubMed2000

The applicability of different PCR-based methods for fetal RHD and K1 genotyping: a prospective study.

The applicability of different PCR-based assays for fetal RHD and K1 genotyping using DNA isolated from uncultured amniotic fluid cells has been tested prospectively: cord blood serotyping served as a control. For RHD genotyping, DNA was amplified with PCRs specific for RHD exon 7, the 3'-non-coding region and intron 4, using standard conditions. The results of these three separate assays were compared to those of a newly-developed multiplex PCR, simultaneously amplifying six regions of RHD. The PCRs analysing the 3'-non-coding region or intron 4 often yielded false-negative results or no results at all. Results of the exon 7 PCR and of the multiplex PCR always corresponded with postnatal serotyping, the multiplex PCR having the advantage of analysing six RHD-specific exons simultaneously. For K1 genotyping, two different PCR-based assays, both analysing the presence of T578C in the KEL gene, were applied. With the first method, a consensus 740-bp product of the KEL gene was amplified and subsequently specifically digested. As we were not able to obtain any PCR product from amniotic fluid DNA, we developed a new K1-specific PCR, amplifying a fragment of 91 bp only in cases of K1-positivity. With this PCR, all K1 genotyping results (n=30) correctly predicted the phenotypes. We conclude that fetal RHD and K1 genotyping can be performed reliably with DNA from uncultured amniotic fluid cells.

Amniotic Fluid↗

PubMed1995

Establishment and evaluation of a multiplex polymerase chain reaction for detection of mycobacteria and specific identification of Mycobacterium tuberculosis complex.

OBJECTIVE: To establish a multiplex polymerase chain reaction for detection of mycobacteria and specific identification of Mycobacterium tuberculosis complex and to evaluate the test in the diagnosis of tuberculosis. DESIGN: Three sets of primers were used to amplify 383 bp, 240 bp and 131 bp DNA fragments from the genes encoding the 65 kDa, MPB64 and the 19 kDa proteins of M. tuberculosis in a single reaction tube. Reaction conditions were optimized with respect to the requirement of DMSO, concentration of MgCl2, annealing and denaturation temperatures and number of amplification cycles. Inhibitory activity in clinical samples was identified by amplifying a 500 bp DNA fragment of the phage lambda along with the mycobacterial targets within the same reaction tube. The multiplex PCR was evaluated in differentiating M. tuberculosis complex from other mycobacteria and in the diagnosis of tuberculosis by testing clinical specimens. RESULTS: Amplification of the 383 bp DNA fragments was specific to the genus Mycobacterium. The 240 bp DNA fragment was amplified from M. tuberculosis complex and M. fortuitum and the 131 bp DNA fragment was amplified from the mycobacteria of M. tuberculosis complex and M. scrofulaceum. All the three bands were amplified only from M. tuberculosis complex. Applicability of the multiplex PCR is demonstrated in differentiating M. tuberculosis complex from other mycobacteria by using standard strains and clinical isolates. The multiplex PCR was also useful in the detection of inhibitory activity and in the identification of M. tuberculosis complex directly in clinical samples. CONCLUSION: The multiplex PCR established in this study could differentiate M. tuberculosis complex from other mycobacteria. This test may also be helpful in the early and specific diagnosis of tuberculosis.

Bacterial Typing Techniques↗

PubMed2001

A successful strategy for preimplantation genetic diagnosis of myotonic dystrophy using multiplex fluorescent PCR.

The most common form of inherited muscular dystrophy in adults is myotonic dystrophy (DM), an autosomal-dominant disease caused by the expansion of an unstable CTG repeat sequence in the 3' untranslated region of the myotonin protein kinase (DMPK) gene. Expanded (mutant) CTG repeat sequences are refractory to conventional PCR, but alleles with a number of repeats within the normal range can be readily amplified and detected. Preimplantation genetic diagnosis (PGD) of DM has been successfully applied. However, a misdiagnosis using the reported protocol was recently documented. Two new PGD protocols for DM have been developed which utilise multiplex fluorescent PCR. Ideally a linked polymorphic marker, APOC2, is amplified in addition to the normal DMPK alleles, thus providing a back-up diagnostic result. However, the two couples reported in the present study were not fully informative at the APOC2 locus and so an unlinked short tandem repeat (STR) marker, D21S1414, was substituted. The highly polymorphic nature of the D21S1414, DMPK and APOC2 loci means that a very simple genetic fingerprint can be generated by analyses of these loci. This allows most DNA contaminants to be detected. Contamination is a significant problem for PGD and is the primary reason for the inclusion of D21S1414 and APOC2 in this protocol. This paper reports the first clinical experience and pregnancies following PGD for DM using a multiplex fluorescent PCR protocol.

Adult↗

PubMed2004

Nucleic acid amplification strategies for DNA microarray-based pathogen detection.

DNA microarray-based screening and diagnostic technologies have long promised comprehensive testing capabilities. However, the potential of these powerful tools has been limited by front-end target-specific nucleic acid amplification. Despite the sensitivity and specificity associated with PCR amplification, the inherent bias and limited throughput of this approach constrain the principal benefits of downstream microarray-based applications, especially for pathogen detection. To begin addressing alternative approaches, we investigated four front-end amplification strategies: random primed, isothermal Klenow fragment-based, phi29 DNA polymerase-based, and multiplex PCR. The utility of each amplification strategy was assessed by hybridizing amplicons to microarrays consisting of 70-mer oligonucleotide probes specific for enterohemorrhagic Escherichia coli O157:H7 and by quantitating their sensitivities for the detection of O157:H7 in laboratory and environmental samples. Although nearly identical levels of hybridization specificity were achieved for each method, multiplex PCR was at least 3 orders of magnitude more sensitive than any individual random amplification approach. However, the use of Klenow-plus-Klenow and phi29 polymerase-plus-Klenow tandem random amplification strategies provided better sensitivities than multiplex PCR. In addition, amplification biases among the five genetic loci tested were 2- to 20-fold for the random approaches, in contrast to >4 orders of magnitude for multiplex PCR. The same random amplification strategies were also able to detect all five diagnostic targets in a spiked environmental water sample that contained a 63-fold excess of contaminating DNA. The results presented here underscore the feasibility of using random amplification approaches and begin to systematically address the versatility of these approaches for unbiased pathogen detection from environmental sources.

Antigens, Bacterial↗

PubMed2005

Rapid identification of virulence genes in enterotoxigenic Escherichia coli isolates associated with diarrhoea in Queensland piggeries.

OBJECTIVE: To identify virulence genes in enterotoxigenic E coli (ETEC) isolates associated with diarrhoea in neonatal, 1 to 3 week-old and weaned pigs in southeast Queensland. DESIGN: Multiplex PCR and serotyping were applied to E coli isolates obtained over a 5-year period (1998-2002) from cases diagnosed at Toowoomba Veterinary Laboratory. PROCEDURE: A total of 126 isolates from 25 different Queensland piggeries were tested for haemolytic activity on 5% sheep blood agar and by multiplex PCR for the presence of five commonly recognised fimbrial (F4, F5, F6, F41 and F18) and three enterotoxin genes (STa, STb, LT). A subset of 62 representative isolates were serotyped by slide agglutination. For comparative purposes, multiplex PCR was also performed on the DNA of 31 ETEC isolates from 9 serotypes originating from piggeries in southern New South Wales. RESULTS: A total of 113 (89.7%) of the isolates from Queensland possessed ETEC virulence genes, including 14 of 15 isolates from neonatal pigs (93.3%), 18 of 23 isolates from 1 to 3 week old pigs (78.3%) and 81 of 88 isolates from weaned pigs (92.1%). F4:STa:STb:LT (serotype O149) was the most prevalent pathotype in neonatal and 1-3 week old pigs and F4:STa:STb:LT (serotype O149) and F18:STa:STb:LT (serotype O141) were most prevalent in weaned pigs. In comparison, isolates obtained from neonatal pigs from New South Wales belonged to a more diverse range of pathotypes and serotypes. CONCLUSION: Multiplex PCR was a rapid and specific method for detecting the presence of ETEC virulence genes in porcine E coli isolates. For isolates obtained from cases of suspected colibacillosis in Queensland, growth of a heavy pure culture of haemolytic E coli was a sensitive prognostic indicator of the presence of ETEC virulence genes in the isolate. ETEC pathotypes and serotypes remained stable in Queensland piggeries over the five-year study period and appear to have changed little over the last three decades.

Animals↗

PubMed2004

A multiplexed PCR-coupled liquid bead array for the simultaneous detection of four biothreat agents.

We have developed a 10-plexed PCR assay coupled to a 12-plexed liquid bead array to rapidly screen environmental samples for B. anthracis, Y. pestis, F. tularensis, and B. melitensis. Highly validated species-specific primer sets were used to simultaneously amplify multiple diagnostic regions unique to each individual pathogen. Resolution of the mix of amplified products was achieved by PCR product hybridization to corresponding probe sequences, attached to unique sets of fluorescent beads. The hybridized beads were processed through a flow cytometer, which detected presence and quantity of each PCR product. The assay was optimized to allow for maximum sensitivity in a multiplexed format. A high-throughput demonstration was performed where 384 simulated environmental samples were spiked with different amounts of B. thuringensis spores and pathogen DNA. The samples were robotically processed to extract DNA and arrayed for multiplexed PCR-liquid bead detection. The assay correctly identified the presence or absence of each pathogen and collected over 3000 individual data points within a single 8-h shift for approximately $4.00 material costs per environmental sample in a 10-plexed assay.

Bacillus anthracis↗