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PubMed2003

Detection of human cytomegalovirus, human herpesvirus type 6 and human herpesvirus type 7 in urine specimens by multiplex PCR.

OBJECTIVES: To develop a sensitive multiplex PCR to detect HCMV, HHV6 and HHV7, to test this PCR on urine specimens sent to the virus diagnostic laboratory and on stored urine samples from HIV-positive patients and their HIV-negative partners and to compare the sensitivity of the multiplex PCR with the diagnostic laboratory's routine service for the detection of HCMV. STUDY DESIGN: Primers specific for each of the three viruses were combined in a multiplex PCR that was then optimised for sensitivity. This PCR was applied prospectively to 413 unselected routine urine specimens over a 1 year period and retrospectively to 258 urine specimens from 63 HIV-positive patients and 10 HIV-negative partners. METHODS: In the prospective study, the multiplex PCR detected 40 specimens positive for HCMV alone, 10 for HHV6, 3 for HHV7 and 3 with a dual infection of HCMV and HHV6. The sensitivity for HCMV was 93.5% by multiplex PCR compared to 28.3% by culture. HHV6 DNA was detected in 6 neonates (2-21 days) and HHV7 DNA in 2 neonates (4 and 20 days). In the retrospective study of HIV patients, HCMV was the most commonly detected virus (55.6%) compared to HHV6 (7.9%) and HHV7 (4.8%). CONCLUSIONS: . The multiplex PCR was significantly more sensitive than non-DNA based procedures for the detection of HCMV. Urine may be a useful non-invasive specimen for the detection of HHV6 and HHV7 and their presence in neonates suggest perinatal transmission or the possibility of in utero infection.

Cytomegalovirus↗

PubMed2002

Influenza virus types and subtypes detection by single step single tube multiplex reverse transcription-polymerase chain reaction (RT-PCR) and agarose gel electrophoresis.

Influenza virus type and subtype specific primers were selected for use in reverse transcription polymerase chain reaction (RT-PCR). The selected primer sets were used in a single step RT-PCR of influenza virus RNA in multiplex format for the detection of virus type and subtypes. Three one step reaction conditions are optimized: (1) multiplex typing only, (2) multiplex subtyping of influenza A, and (3) multiplex typing and subtyping simultaneously. RNA from strains of influenza virus type A of subtypes H1N1, H3N2 and H5N1 and influenza virus type B was used for amplification and detection. The amplified DNA fragments were size analyzed for the presence of specific type and subtypes target DNA by agarose gel electrophoresis. The sensitivity of detection was about 0.01 TCID(50) for both influenza type A and B when amplification was for typing alone. In multiplex subtyping or multiplex typing and subtyping simultaneously, the sensitivities for types and subtypes specific bands were 0.01-0.1 TCID (50) in the tested volume.

Electrophoresis, Agar Gel↗

PubMed1998

A multiplex PCR assay for the simultaneous detection of human herpesvirus 6 and human herpesvirus 7, with typing of HHV-6 by enzyme cleavage of PCR products.

A multiplex polymerase chain reaction (PCR) method was developed for the simultaneous detection of human herpesviruses 6 and 7 (HHV-6; HHV-7) in clinical samples, using primers which amplify a section of the HHV-6 U67 gene and the HHV-7 homologue of the HHV-6 U42 gene. Comparison of the multiplex assay with the respective single PCR assays, using cloned HHV-6 and HHV-7 sequences as targets for amplification, showed equivalent sensitivity and specificity for the assays. To demonstrate the use of multiplex PCR for the analysis of clinical samples, serum and saliva from infants were analysed using this technique. The results showed that a clear distinction can be made between the amplicons of HHV-6 and HHV-7, without loss of sensitivity or specificity. There was complete concordance between the respective single PCR assays, and the multiplex PCR. HHV-6 amplicons derived from the multiplex PCR analysis were typed by differential AvaII restriction endonuclease digestion, in which HHV-6 variant A amplicons are cleaved but those of variant B remain undigested. These results were compared to HHV-6 variant typing by an established method, the results of which showed complete concordance between assays. It is proposed that this multiplex assay, where HHV-6 positive samples may be typed directly from the reaction products, is an efficient and cost-effective approach to the analysis of large numbers of samples to determine the epidemiological importance of HHV-6 and HHV-7.

Child↗

PubMed2005

MuPlex: multi-objective multiplex PCR assay design.

We have developed a web-enabled system called MuPlex that aids researchers in the design of multiplex PCR assays. Multiplex PCR is a key technology for an endless list of applications, including detecting infectious microorganisms, whole-genome sequencing and closure, forensic analysis and for enabling flexible yet low-cost genotyping. However, the design of a multiplex PCR assays is computationally challenging because it involves tradeoffs among competing objectives, and extensive computational analysis is required in order to screen out primer-pair cross interactions. With MuPlex, users specify a set of DNA sequences along with primer selection criteria, interaction parameters and the target multiplexing level. MuPlex designs a set of multiplex PCR assays designed to cover as many of the input sequences as possible. MuPlex provides multiple solution alternatives that reveal tradeoffs among competing objectives. MuPlex is uniquely designed for large-scale multiplex PCR assay design in an automated high-throughput environment, where high coverage of potentially thousands of single nucleotide polymorphisms is required. The server is available at http://genomics14.bu.edu:8080/MuPlex/MuPlex.html.

Algorithms↗

PubMed2001

Fluorescent multiplex polymerase chain reaction analysis of four genes associated with inpaired fibrinolysis and myocardial infarction.

Multiplex polymerase chain reaction (PCR) allows for the simultaneous amplification of several genes, thereby optimizing the use of reagents and decreasing personnel time. Multiplex PCR was used to amplify four genes in one PCR reaction, demonstrating the advantage of multiplex PCR for our study since it allowed us to amplify four separate genes using only 1 microl DNA, thus maximizing the use of study DNA. As compared with conventional multiplex PCR analysis with ethidium bromide, incorporating fluorescence-labeled primers into multiplex PCR reactions facilitated accurate, simultaneous analysis of many DNA fragments within one base discrimination. We have used this fluorescence methodology to analyze polymorphisms associated with either impaired fibrinolysis or myocardial infarction. These include the angiotensin converting enzyme insertion/deletion (I/D) polymorphism in intron 16 of the DCP1 gene, the Alu I/D polymorphism of the tissue plasminogen activator-25 locus in intron 8, the plasminogen activator inhibitor 4G/5G repeat polymorphism, and the variable number tandem repeat of the endothelial cell nitric oxide synthase gene, all characterized by an insertion, deletion, or repeat. The amplified products were diluted 1 : 60 and analyzed on the ABI PRISM 310 Genetic Analyzer using GeneScan software. With this method, we were able to amplify four genes using 75% less reagents and personnel time, thus demonstrating the benefit of multiplex PCR and fluorescence technology.

Alleles↗

PubMed2001

Familial lupus erythematosus. Clinical and immunologic features of 125 multiplex families.

Evidence for a genetic susceptibility to systemic lupus erythematosus (SLE) in humans is based on the high concordance rate observed in identical twins and on the relatively high incidence of familial cases. Although recent genetic studies have lead to significant advances in the identification of new susceptibility genes in SLE, no large clinico-pathologic study of familial SLE has been reported to date. In the present study, we describe the main clinical and immunologic features of 125 lupus multiplex families including at least 2 cases of SLE and/or discoid lupus erythematosus (DLE), recruited through a French national survey starting in July 1997. Medical records of all affected members were reviewed by the same investigator, all available family members were interviewed using the same standardized procedure, and blood was drawn for autoantibodies typing. Clinical and immunologic features of 90 probands from multiplex SLE families were compared with those of 100 sporadic SLE patients sharing the same French Caucasian origin. The 125 lupus multiplex families included 282 affected members (2.3 patients per family); of the 125 families, 96 were of French Caucasian origin. One hundred multiplex families included 2 affected relatives, while 25 included 3 or more affected individuals. The relationship between affected members was sibs (45%), parent-offspring (31%), and second-degree (24%). An autosomal dominant mode of inheritance was strongly suggested in 1 extended pedigree with 6 clinically affected members, and a recessive pattern was suspected in 5 other families. No obvious mode of inheritance could be suspected in most of the remainder. Among French Caucasians, sex ratio, mean age at onset, and clinical and biologic SLE-related manifestations were not significantly different in multiplex compared with sporadic SLE cases. The analysis of these 125 multiplex families suggests a genetic heterogeneity that should be considered for ongoing genomic screening.

Age of Onset↗

PubMed2005

Rapid differentiation and in situ detection of 16 sourdough lactobacillus species by multiplex PCR.

A two-step multiplex PCR-based method was designed for the rapid detection of 16 species of lactobacilli known to be commonly present in sourdough. The first step of multiplex PCR was developed with a mixture of group-specific primers, while the second step included three multiplex PCR assays with a mixture of species-specific primers. Primers were derived from sequences that specify the 16S rRNA, the 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene. The primer pairs designed were shown to exclusively amplify the targeted rrn operon fragment of the corresponding species. Due to the reliability of simultaneously identifying Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum, a previously described multiplex PCR method employing recA gene-derived primers was included in the multiplex PCR system. The combination of a newly developed, quick bacterial DNA extraction method from sourdough and this multiplex PCR assay allows the rapid in situ detection of several sourdough-associated lactobacilli, including the recently described species Lactobacillus rossii, and thus represents a very useful alternative to culture-based methodologies.

Bacterial Typing Techniques↗

PubMed2004

Evaluation of multiplexed fluorescent microsphere immunoassay for detection of autoantibodies to nuclear antigens.

Antibodies to extractable nuclear antigens (ENA) are found in a variety of collagen vascular diseases. Determining the individual specificities of these antibodies is extremely useful in establishing the disease diagnosis and in some cases the prognosis. With a multiplexed fluorescent microsphere immunoassay, reactivity to five of the most diagnostically useful ENA was measured in 249 serum samples, including samples from 56 patients previously documented to have systemic lupus erythematosus (SLE). Results of the multiplexed assay were compared to results from established ENA enzyme-linked immunosorbent assays (ELISAs), and the agreement, sensitivity, and specificity, respectively, for the five ENA evaluated were as follows: SSA, 99.1, 100.0, and 98.8%; SSB, 98.6, 88.9, and 99.5%; Sm, 97.6, 95.8, and 97.9%; RNP, 97.2, 92.7, and 98.8%; Scl-70, 93.6, 50.0, and 99.0%. In the 56 confirmed SLE patients, the frequency of significant concentrations of autoantibodies with the multiplexed assay was 21.4% for SSA, 7.1% for SSB, 10.7% for Sm, 32.1% for RNP, and 0% for Scl-70. The new flow cytometric bead-based multiplexed assay showed excellent correlation with the well-established single-analyte ELISA methods for four of five the ENA markers investigated in this study. The most notable discrepancies between the two assays were for the Scl-70 antigen, which was most often resolved in favor of the multiplexed assay. Our studies show that the multiplexed microsphere-based immunoassay is a sensitive and specific method for the detection and semiquantitation of ENA antibodies in human sera.

Antibodies, Antinuclear↗

PubMed2001

Laboratory diagnosis of common herpesvirus infections of the central nervous system by a multiplex PCR assay.

A sensitive multiplex PCR assay for single-tube amplification that detects simultaneous herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), human cytomegalovirus (CMV), and Epstein-Barr virus (EBV) is reported with particular emphasis on how the method was optimized and carried out and its sensitivity was compared to previously described assays. The assay has been used on a limited number of clinical samples and must be thoroughly evaluated in the clinical context. A total of 86 cerebrospinal fluid (CSF) specimens from patients which had the clinical symptoms of encephalitis, meningitis or meningoencephalitis were included in this study. The sensitivity of the multiplex PCR was determined to be 0.01 and 0.03 50% tissue culture infective doses/the reciprocal of the highest dilution positive by PCR for HSV-1 and HSV-2 respectively, whereas for VZV, CMV and EBV, 14, 18, and 160 ag of genomic DNA were detected corresponding to 48, 66, and 840 genome copies respectively. Overall, 9 (10.3%) of the CSF samples tested were positive in the multiplex PCR. HSV-1 was detected in three patients (3.5%) with encephalitis, VZV was detected in four patients (4.6%) with meningitis, HSV-2 was detected in one neonate (1.16%), and CMV was also detected in one neonate (1.16%). None of the samples tested was positive for the EBV genome. None of the nine positive CSF samples presented herpesvirus coinfection in the central nervous system. Failure of DNA extraction or failure to remove any inhibitors of DNA amplification from CSF samples was avoided by the inclusion in the present multiplex PCR assay of alpha-tubulin primers. The present multiplex PCR assay detects simultaneously five different herpesviruses and sample suitability for PCR in a single amplification round of 40 cycles with an excellent sensitivity and can, therefore, provide an early, rapid, reliable noninvasive diagnostic tool allowing the application of antiviral therapy on the basis of a specific viral diagnosis. The results of this preliminary study should prompt a more exhaustive analysis of the clinical value of the present multiplex PCR assay.

Central Nervous System Viral Diseases↗

PubMed2002

Evaluation of a multiplex reverse transcription-polymerase chain reaction assay for subtyping hemagglutinin genes 1 and 3 of swine influenza type A virus in clinical samples.

A multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to detect and identify subtypes of hemagglutinin (H) 1 and H3 swine influenza virus (SIV). Two oligonucleotide primer sets were prepared using published sequence data for H1N1 and H3N2. The PCR products with unique size characteristics of each subtype were sequenced, and the sequences were confirmed to be subtype specific for H gene 1 or 3. These primer sets did not amplify when RT-PCR assay was performed for genomic DNA or RNA from other common swine pathogens. The RT-PCR assay was able to detect viral RNA up to 1 tissue culture infective dose of reference SIV H1N1 or H3N2. The multiplex RT-PCR was applied to 30 SIV isolates subtyped by hemagglutination inhibition (HI) test. Forty-three positive and 20 negative swine field samples for SIV by virus isolation were also tested. Of these 73 SIV-positive samples tested, H1 and H3 were identified in 38 and 28 samples by the multiplex RT-PCR, respectively. The remaining 7 samples were positive for both H1 and H3 genes. No positive reaction was found for all 20 SIV-negative field samples. Subsequently, 235 random field samples from pigs with respiratory problems were tested by the multiplex RT-PCR, and 26 and 13 samples were found to be positive for H1 and H3, respectively. Results of these multiplex RT-PCR were comparable with those of the HI test. These results suggest that multiplex RT-PCR can be a useful test for detection and subtyping of SIV in clinical samples.

Animals↗

PubMed2006

Statistical thermodynamics and kinetics of DNA multiplex hybridization reactions.

A general analytical description of the equilibrium and reaction kinetics of DNA multiplex hybridization has been developed. In this approach, multiplex hybridization is considered to be a competitive multichannel reaction process: a system wherein many species can react both specifically and nonspecifically with one another. General equations are presented that can consider equilibrium and kinetic models of multiplex hybridization systems comprised, in principle, of any number of targets and probes. Numerical solutions to these systems for both equilibrium and kinetic behaviors are provided. Practical examples demonstrate clear differences between results obtained from more common simplex methods, in which individual hybridization reactions are considered to occur in isolation; and multiplex hybridization, where desired and competitive cross-hybrid reactions between all possible pairs of strands are considered. In addition, sensitivities of the hybridization process of the perfect match duplex, to temperature, target concentration, and existence of sequence homology with other strands, are examined. This general approach also considers explicit sequence-dependent interactions between targets and probes involved in the reactions. Sequence-dependent stabilities of all perfect match and mismatch duplex complexes are explicitly considered and effects of relative stability of cross-hybrid complexes are also explored. Results reveal several interdependent factors that strongly influence DNA multiplex hybridization behavior. These include: relative concentrations of all probes and targets; relative thermodynamic stability of all perfect match and mismatch complexes; sensitivity to temperature, particularly for mismatches; and amount of sequence homology shared by the probe and target strands in the multiplex mix.

Base Sequence↗

PubMed1996

Multiplex-PCR-based single-strand conformation polymorphism protocol for simultaneous analysis of up to five fragments of the low-density-lipoprotein receptor gene.

Single-strand conformation polymorphism has become a screening method for the detection of mutations in different genes. For analysis of the promotor region and the coding sequence of the low-density-lipoprotein receptor gene by standard protocols, 21 radiolabeled PCRs and electrophoreses have to be performed. To accelerate this procedure, we developed a nonradioactive multiplex approach of the single-strand conformation polymorphism analysis. Multiplex PCRs were established, each resulting in the amplification of 4 or 5 fragments of this gene. The heat-denatured, single-stranded multiplex-PCR products were electrophoresed, blotted on a nylon membrane and visualized using a chemiluminescence detection system. The simultaneously amplified fragments were clearly resolved by their different mobility on the gel. Comparing the pattern of bands of each separately amplified PCR product and the multiplex-PCR products allowed identification of each band as one exon, part of an exon or the promotor region of the gene. To determine the sensitivity of this method, the low-density-lipoprotein receptor gene of 11 patients with 11 different mutations was analyzed. All mutations could be identified in the multiplex reactions. We conclude that a multiplex-PCR-based, single-strand conformation polymorphism protocol is much faster but equally sensitive compared to standard protocols.

Codon↗

PubMed2005

Evaluation of a multiplex polymerase chain reaction assay for simultaneous detection of Rhodococcus equi and the vapA gene.

OBJECTIVE: To evaluate sensitivity and specificity of a multiplex polymerase chain reaction (PCR) assay for simultaneous detection of Rhodococcus equi and differentiation of strains that contain the virulence-associated gene (vapA) from strains that do not. SAMPLE POPULATION: 187 isolates of R equi from equine and nonequine tissue and environmental specimens and 27 isolates of bacterial species genetically or morphologically similar to R equi. PROCEDURE: The multiplex PCR assay included 3 gene targets: a universal 311-bp bacterial 16S ribosomal RNA amplicon (positive internal control), a 959-bp R equi-specific target in the cholesterol oxidase gene (choE), and a 564-bp amplicon of the vapA gene. Duplicate multiplex PCR assays for these targets and confirmatory singleplex PCR assays for vapA and choE were performed for each R equi isolate. An additional PCR assay was used to examine isolates for the vapB gene. RESULTS: Results of duplicate multiplex and singleplex PCR assays were correlated in all instances, revealing high specificity and reliability (reproducibility) of the vapA multiplex assay. Of the pulmonary isolates from horses with suspected R equi pneumonia, 97.4% (76/78) yielded positive results for vapA. Seven of 50 (14%) human isolates of R equi yielded positive results for vapA. Six human R equi isolates and 1 porcine isolate yielded positive results for vapB. No isolates with vapA and vapB genes were detected. CONCLUSIONS AND CLINICAL RELEVANCE: The multiplex PCR assay is a sensitive and specific method for simultaneous confirmation of species identity and detection of the vapA gene. The assay appeared to be a useful tool for microbiologic and epidemiologic diagnosis and research.

Actinomycetales Infections↗

PubMed2001

Comparison of culture, multiplex, and 5' nuclease polymerase chain reaction assays for the rapid detection of Yersinia enterocolitica in swine and pork products.

Bacteriological culture was compared with multiplex and fluorogenic (TaqMan) polymerase chain reaction (PCR) assays for the detection of attachment invasion locus (ail)-bearing Yersinia enterocolitica in market weight swine, chitterlings, and ground pork. The TaqMan assay detected 1 pg of purified Y. enterocolitica DNA, whereas conventional gel-based PCR detected I ng of the same. The presence of ail-bearing Y. enterocolitica was tested in pork and feces artificially inoculated with Y. enterocolitica strain NADC 5561. The sensitivity limits of culture, multiplex, and TaqMan PCR assays were 4 x 10(3), 4 x 10(2), and 0.4 CFU/g, respectively, for the artificially inoculated pork. The sensitivity limits were 4 x 10(2), 4 x 10(2), and 0.4 CFU/g, respectively, for feces after a 48-h enrichment in a Yersinia selective broth. By the culture method, Y. enterocolitica was not detected in any of the swine specimens (n = 2,403) examined. By contrast, it was detected in 48 (2%) of the swine samples screened using the multiplex PCR and in 656 (27.2%) of these samples using the TaqMan assay. Using the culture method, Y. enterocolitica was detected in 8% of chitterling samples (n = 350) and in none of the ground pork samples (n = 350). It was identified in 27% of the chitterling samples using multiplex PCR and in 79% of these samples using the TaqMan assay. Ten percent of the ground pork samples contained Y. enterocolitica, as determined by the multiplex PCR, and 38% based on the TaqMan assay. The results suggest that pork products harbor more ail-bearing Y. enterocolitica than selected organs of freshly slaughtered hogs and that the TaqMan assay is more sensitive than either the multiplex PCR or traditional culture methods.

Animals↗

PubMed2000

Detection of adenoviruses and enteroviruses in tap water and river water by reverse transcription multiplex PCR.

A reverse transcription (RT) multiplex polymerase chain reaction (PCR) assay was developed to simultaneously detect adenoviruses and enteroviruses, both of which have attracted much attention as molecular indices of viral pollution in environmental samples. The method involves a reverse transcription step, followed by a multiplex nested PCR in which the combination of primers amplifies cDNA from enteroviruses and adenoviruses. The sensitivity of this assay was found to be similar to that of each monoplex PCR or RT-PCR assay, and to be consistent regardless of relative concentrations of adenoviruses and enteroviruses. To assess suitability and environmental application of the RT multiplex PCR assay, a total of 12 river water samples and 4 tap water samples were analyzed by RT multiplex PCR, each monoplex PCR or RT-PCR, and cell culture assay on the Buffalo Green Monkey kidney cell line. The sensitivity of the RT multiplex PCR was also found to be similar to that of each monoplex PCR in environmental samples. This suggests the RT multiplex PCR assay could be applied to the routine monitoring of viral pollution in environmental waters.

Adenovirus Infections, Human↗

PubMed2001

Nested multiplex polymerase chain reaction for the diagnosis of cutaneous herpes simplex and herpes zoster infections and a comparison with electronmicroscopy.

Herpes simplex virus (HSV) and varicella zoster virus (VZV) are common causes of cutaneous and mucocutaneous vesicular eruptions. Laboratory diagnostic techniques include Tzanck smears, electronmicroscopy, antigen detection and viral culture. This paper describes a nested multiplex polymerase chain reaction with respective sensitivities of 0.0001, 0.01 and 0.1 TCID50 for VZV, HSV-1 and HSV-2. The assay was used in (a) a salvage capacity for slides already processed for electronmicroscopy, and (b) as a front-line assay for prospectively processed specimens. Sixty-two glass slides with vesicle lymph/scrapings from 58 patients with suspected cutaneous herpetic lesions were examined. The clinical presentations were described as atypical/not specified (24), VZV (20) or HSV (18), and involved eruptions from diverse anatomical sites, including the genitalia. Of the 62 specimens, 6 and 38 were positive by electronmicroscopy and multiplex PCR respectively, giving a comparative sensitivity of 16% for electronmicroscopy. Nested multiplex PCR identified 15 VZV and 20 HSV-1 infections. Where the clinical details indicated either HSV or VZV (38/62), nested multiplex PCR was statistically likely to be reactive (26/38 vs. 9/24) (chi2 P = 0.000004) whereas electronmicroscopy was not (4/38 vs. 2/24) (chi2 P= 0.77). Where the clinical details indicated VZV (20/62) or HSV (18/62), nested multiplex PCR was statistically more likely to confirm VZV (10/20 vs. 5/42) (chi2 P= 0.001) or HSV (9/18 vs. 11/44) (chi2 P = 0.05) respectively. Two suspected HSV and 6 suspected VZV infections were shown to be VZV and HSV respectively by nested multiplex PCR.

Adolescent↗

PubMed1999

[Establishment of a multiplex PCR system to detect plasmodium].

AIM: To establish a simple, rapid and practical multiplex PCR system to detect Plasmodium vivax(P. v) and Plasmodium falciparum(P. f). METHODS: A common upper primer S1 and two species-specific lower primers of P. v and P. f, S2, S3 were designed according to the sequences of the small subunit ribosomal DNA (SSUrDNA) fragments of the two Plasmodium species. Using these three oligonucleotide primers, the two-temperature-point multiplex PCR system was established and applied to detect P. v and P. f in the stock blood samples of clinically confirmed patients. RESULTS: DNA fragments of about 705 and 575 base pairs were successfully amplified by multiplex PCR from the genomic DNA of P. v and P. f, but no fragments were obtained from that of P. knowlesi, P. cynomolgi and blood of healthy persons. By means of restriction endonuclease digestion, the amplified fragments were confirmed to be the SSUrDNA fragments of P. v and P. f as expected. This method was successfully used in detecting parasitemia 2-10 parasite/microliter whole blood. Of 104 samples tested by this system, 81 were coincident with microscopic examination. The multiplex PCR system also found 17 samples of mixed infection, which were not detected microscopically. Another 2 samples detected as P. v by microscopic examination were verified to be P. f infection by the multiplex PCR. CONCLUSION: The ease of operation together with high sensitivity and specificity, particularly the sensitive detection for mixed infection in a single run of amplification suggests that the multiplex PCR system might be a useful tool for malaria diagnosis.

Animals↗

PubMed2004

Validation and implementation of the PowerPlex 16 BIO System STR multiplex for forensic casework.

The PowerPlex 16 BIO multiplex short tandem repeat (STR) system contains the 13 CODIS loci (FGA, TPOX, D8S1179, vWA, D18S51, D21S11, TH01, D3S1358, CSF1PO, D16S539, D7S820, D13S317, and DS5S818), plus two pentanucleotide repeat loci (Penta D and Penta E) and the sex-identifying locus. Amelogenin. The PowerPlex 16 BIO System is optimized for use with the Hitachi FMBIO gel imaging systems. A consortium of seven independent laboratories collaborated to perform the studies defined by the FBI standards for performing a developmental validation, including the evaluation of sample concordance, percent stutter determination, nonprobative casework, precision, sensitivity, mixture determination, effect of substrates, the impact of environmental insults, and species specificity. All samples tested for concordance were consistent except for one sample from the Virginia Division of Forensic Science database that displayed discordance at D13S317, a locus whose primer sequence was altered. Stutter values were comparable to those of other STR multiplex systems, the precision was comparable to other multiplexes analyzed by gel electrophoresis, the DNA profiles were unchanged by the substrate upon which the blood samples were placed, and the nonprobative casework samples re-typed for the PowerPlex 16 BIO System were consistent with previous typing results. When greater than 0.125 ng of DNA was placed into the PowerPlex 16 BIO System amplification reaction, a full profile was generated by all laboratories. The mixture study results were comparable to those reported for other multiplex systems, the environmental study demonstrated a loss of larger molecular weight loci when samples were incubated at elevated temperatures for a prolonged period of time, and the only notable cross species hybridization was observed with primate DNA samples. This extensive validation work performed demonstrates that the PowerPlex 16 BIO System provides STR data of a quality comparable with other PowerPlex STR multiplex kits as well as other widely used STR multiplexes and is thus suitable for evidentiary casework analysis as well as database sample profiling.

Alleles↗