Multiplex Polymerase Chain Reaction Reactions Research Articles

Development of microsatellite markers for the invasive mosquito Aedes koreicus

BackgroundAedes koreicus is a mosquito species native to East Asia which has recently invaded several countries in Europe. In Italy, this mosquito was first detected in the North-East in 2011 and is now widely distributed in the entire northern part of the country. The development of specific genetic markers, such as microsatellites, is necessary to uncover the dispersal routes of this mosquito from its native areas and, eventually, to plan future control interventions.MethodsAvailable raw sequences of genomic DNA of Ae. koreicus were screened in silico using BLASTn to identify possible microsatellite-containing sequences. Specific primer pairs were then designed, and their efficiency was determined through polymerase chain reaction (PCR) on 32 individuals of Ae. koreicus collected in Italy. PCR conditions were optimised in three multiplex reactions. Genotyping of individual mosquitoes was performed on both single and multiplex PCR reactions. Finally, analysis of intra-population variation was performed to assess the level of polymorphism of the markers.ResultsMosquito genotyping provided consistent results in both single and multiplex reactions. Out of the 31 microsatellite markers identified in the Ae. koreicus genome raw sequences, 11 were polymorphic in the examined mosquito samples.ConclusionsThe results show that the 11 microsatellite markers developed here hold potential for investigating the genetic structure of Ae. koreicus populations. These markers could thus represent a novel and useful tool to infer the routes of invasion of this mosquito species into Europe and other non-native areas.Graphical

Development and Implementation of Multiplex TaqMan Array Cards for Specimen Testing at Child Health and Mortality Prevention Surveillance Site Laboratories.

Child Health and Mortality Prevention Surveillance (CHAMPS) laboratories are employing a variety of laboratory methods to identify infectious agents contributing to deaths of children <5 years old and stillbirths in sub-Saharan Africa and South Asia. In support of this long-term objective, our team developed TaqMan Array Cards (TACs) for testing postmortem specimens (blood, cerebrospinal fluid, lung tissue, respiratory tract swabs, and rectal swabs) for >100 real-time polymerase chain reaction (PCR) targets in total (30–45 per card depending on configuration). Multipathogen panels were configured by syndrome and customized to include pathogens of significance in young children within the regions where CHAMPS is conducted, including bacteria (57 targets covering 30 genera), viruses (48 targets covering 40 viruses), parasites (8 targets covering 8 organisms), and fungi (3 targets covering 3 organisms). The development and application of multiplex real-time PCR reactions to the TAC microfluidic platform increased the number of targets in each panel while maintaining assay efficiency and replicates for heightened sensitivity. These advances represent a substantial improvement in the utility of this technology for infectious disease diagnostics and surveillance. We optimized all aspects of the CHAMPS molecular laboratory testing workflow including nucleic acid extraction, quality assurance, and data management to ensure comprehensive molecular testing of specimens and high-quality data. Here we describe the development and implementation of multiplex TACs and associated laboratory protocols for specimen processing, testing, and data management at CHAMPS site laboratories.

DNA Barcode Quantification As a Robust Tool for Measuring Mixing Ratios in Two-Component Systems.

For many manufacturing processes, correct mixing compositions are crucial to guarantee product quality. However, the analysis of mixing ratios based on component balances can be challenging and requires extensive infrastructure. DNA barcodes have been previously proposed as low-cost markers for product authenticity, and we show here that the quantification of such barcodes via a quantitative real-time polymerase chain reaction (PCR) enables the determination of mixing ratios in a range of liquid and polymeric products. To enable the distribution of the DNA within the various matrixes, the biochemical is encapsulated in silica nanoparticles and distributed within the matrix of the raw material. If both raw materials of a two-component mixture contain such barcodes, the composition of the mixture can be determined from the relative concentration of the barcodes via multiplex PCR reactions, irrespective of the sampling volume and for a wide range of initial barcode concentrations (10 ppm to 10 ppb). As an application example, we use the barcodes to determine the mixing ratios of cross-linked and multicomponent polysilicon products.

Multiplex polymerase chain reaction detection of Curvularia lunata, Bipolaris maydis, and Aureobasidium zeae in infected maize leaf tissues

Multiplex polymerase chain reaction (PCR) is a method for simultaneous identification and detection of multiple pathogenic fungi, however, its complexity limits its application. To simplify the protocol and to improve the effectiveness, three-level designs for six factors (three primers, Taq DNA polymerase, dNTP, Mg2+ ) were constructed to optimize the multiplex PCR system by using the orthogonal design method and the annealing temperature of the PCR reactions was also optimized. Finally, a multiplex PCR system for the simultaneous detection of these three pathogens of maize was successfully established. The reaction volume was 25 μl and the annealing temperature was 57℃. The optimal conditions for multiplex PCR reaction contained 0.48 μmol/L Cl-1/Cl-2, 0.72 μmol/L Bm-1/Bm-2, 0.24 μmol/L Az-1/Az-2, 1.5 U polymerase, 0.35 mmol/L dNTP, and 1.25 mmol/L MgCl2 . The multiplex PCR system can detect Curvularia lunata, Bipolaris maydis, and Aureobasidium zeae in infected plant tissues rapidly with the sensitivity at 10 pg DNA/μl.

A fast multiplex real-time PCR assay for simultaneous detection of pork, chicken, and beef in commercial processed meat products

As rapid and efficient methods for species identification were required, we developed a fast multiplex real-time polymerase chain reaction (PCR) assay based on TaqMan® probes to simultaneously detect pork, chicken, and beef in processed meat samples within 40 min. For multiplex PCR reactions, the 5’ ends of the pork-, chicken-, and beef-specific probes were labeled with FAM, VIC, and NED, respectively. The specificity of this assay was evaluated using 20 animal species and no cross-contamination was observed. The absolute detection limit was 0.1 pg of DNA extracted for all three target species, and the limit of detection (LOD) of reference meat mixtures was determined to be 0.5%. The optimized method was applied to 66 commercial processed products and specific targets were successfully amplified in under 40 min. Therefore, the fast multiple real-time PCR method can be utilized for easily and rapidly monitoring the presence of three meat species in various processed meat products.

Use of Multiplex Polymerase Chain Reaction for Detection of High-Risk Human Papillomavirus Genotypes in Women Attending Routine Cervical Cancer Screening in Harare

Background/Aims: In Zimbabwe, cervical cancer is screened through cytology and visual inspection with acetic acid and cervicography (VIAC). The effectiveness of these methods can be increased if complemented by a human papillomavirus DNA detection tool since most cervical cancer cases are caused by persistent infection with high-risk human papillomavirus (HR-HPV) genotypes. Moreover, the possibility of multiple-genotype HR-HPV infections warrants the need for HPV detection tools with the capacity to detect both single and multiple infections. The aim of this study was to detect HR-HPV genotypes (HPV 16, 18, 31, 33, 35, 45, 51, 52, 56, and 58), using multiplex polymerase chain reaction (PCR), in stored cervicovaginal swabs from both HIV-positive and HIV-negative women reporting for routine cervical cancer screening. Methodology: Stored cervicovaginal swabs from sexually active women who underwent VIAC at the Parirenyatwa Referral Hospital in Harare, Zimbabwe, between February and April 2015 and had received HIV counselling and testing were genotyped for the selected 10 HR-HPV genotypes using in-house multiplex PCR. The results from the multiplex PCR were compared to those previously obtained when the same samples were HPV genotyped with next-generation sequencing (NGS) on an MiSeq platform (Illumina; USA). Results: A total of 136 women were recruited and all 10 HR-HPV genotypes were detected. Quality control failed in 3 of the 136 swabs during the multiplex PCR reactions. The prevalence of HR-HPV genotypes in the study subjects was 53% (70/133). HIV-infected women were 1.67 times more likely to be infected with HR-HPV than were HIV-negative women (OR 1.67; p = 0.17). Of the 70 HR-HPV-positive cases, 37% (26/70) had multiple HR-HPV infections, and the majority of them were HIV infected. HIV-infected women were 1.86 times more likely to have multiple HR-HPV infections than HIV-negative women (OR 1.86; p = 0.20). Multiplex PCR and NGS had an almost perfect concordance rate in ­HR-HPV detection (κ = 0.960), with only 3 discordant cases (negative with NGS and positive for HPV16 with multiplex PCR). Conclusion: Multiplex PCR can detect HR-HPV genotypes that are common in Zimbabwe and could be used to detect HR-HPV genotypes from women attending cervical cancer screening programs at the Parirenyatwa VIAC clinic in Harare.

Comparative Analysis of Verification Parameters of Event-Specific Methods in GMO Maize

The second most dominant genetically modified (GM) crop is maize. Increasing number of GM maize events puts significant pressure on GMO testing laboratories to achieve the level of competence necessary to fulfill legal requirements. In the European Union, Polymerase Chain Reaction (PCR) is the method of choice for identification and quantification of GMOs. We performed verification of validated methods for identification of four GM maize events. Additionaly we aimed to explore the option of designing a method for simultaneous detection of these events in a multiplex PCR reaction. DNA was extracted from certified reference materials (CRM) using validated CTAB extraction protocol. Concentration of DNA was measured using Qubit dsDNA Broad Range Assay. Amplification of taxon specific marker for maize and all event-specific methods was performed according to the JRC Compendium of Reference Methods for GMO Analysis. Absolute limit of detection (LODabs) was determined for taxon specific and four event specific RealTime PCR based methods. DNA extracted from CRMs showed sufficient concentration for downstream analyses and preparation of dilutions for determination of LODabs. Determined LODabs for all tested methods meet acceptance criteria. As expected, the methods performance with respect to the repeatability and precision decline with the decrease in concentration of the target. Event-specific GA21 and NK603 methods show high Ct values at the determined LODabs. However, by adjusting the concentrations of primers and probes sensitivity of these two methods should be improved. Considering that the amplicons for all five methods are quite short (&lt;120 bp) optimization of multiplex reaction conditions for simultaneous amplification should be feasible.

Development and validation of a Multiplex-PCR tool for the detection of Vibrio alginolyticus, Vibrio anguillarum and Vibrio harveyi in fish

Development and validation of a Multiplex-PCR tool for the detection of Vibrio alginolyticus, Vibrio anguillarum and Vibrio harveyi in fish

Simultaneous Detection of 13 Key Bacterial Respiratory Pathogens by Combination of Multiplex PCR and Capillary Electrophoresis

ObjectiveLower respiratory tract infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory bacteria. To compensate for the limits of current respiratory bacteria detection methods, we developed a combination of multiplex polymerase chain reaction (PCR) and capillary electrophoresis (MPCE) assay to detect thirteen bacterial pathogens responsible for lower respiratory tract infections, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Legionella spp., Bordetella pertussis, Mycobacterium tuberculosis complex, Corynebacterium diphtheriae, and Streptococcus pyogenes. MethodsThree multiplex PCR reactions were built, and the products were analyzed by capillary electrophoresis using the high-throughput DNA analyzer. The specificity of the MPCE assay was examined and the detection limit was evaluated using DNA samples from each bacterial strain and the simulative samples of each strain. This assay was further evaluated using 152 clinical specimens and compared with real-time PCR reactions. For this assay, three nested-multiplex-PCRs were used to detect these clinical specimens. ResultsThe detection limits of the MPCE assay for the 13 pathogens were very low and ranged from 10−7 to 10−2 ng/μL. Furthermore, analysis of the 152 clinical specimens yielded a specificity ranging from 96.5%-100.0%, and a sensitivity of 100.0% for the 13 pathogens. ConclusionThis study revealed that the MPCE assay is a rapid, reliable, and high-throughput method with high specificity and sensitivity. This assay has great potential in the molecular epidemiological survey of respiratory pathogens.

Development of a new multiplex-PCR tool for the simultaneous detection of the fish pathogens Vibrio alginolyticus, Vibrio anguillarum, Vibrio harveyi and Edwardsiella tarda

Disease assessment and management in cultured aquatic animals is a major concern in commercial aquaculture. Disease outbreaks have direct effects on fish production, causing serious economic losses in this industry. This can be overcome by early detection through molecular high sensitivity tools such as PCR (polymerase chain reaction). One of the most critical steps in the study of bacterial fish diseases is the precise identification of the infectious agent. Considering the damage that some bacteria can cause to fish and humans, the development of a rapid detection method for the four target species that demonstrates to be simple, accurate and low cost is an essential step for the prevention and early treatment of these diseases. Edwardsiella tarda (ACC 36.1), Vibrio harveyi (DSM 19023), Vibrio anguillarum (AQV 55.1) and Vibrio alginolyticus (CECT 521) were selected as targets of a multiplex PCR tool. The multiplex PCR reactions were performed in various reaction conditions, including different annealing temperatures (between 49 °C and 55 °C) and changes in the MgCl2 concentration from 2 mM to 8 mM. Best results were obtained at 51 °C and MgCl2 concentration from 4 mM to 6 mM. Primers were tested using purified DNA from the respective bacterial strains, yielding bands at expected sizes, which can be identified on agarose gels with clearness and without overlapping sizes. Results indicate that this multiplex PCR tool is suitable for the detection of target pathogens and may, in the future, have extended practical application in commercial aquaculture.

Diversity of CRISPR loci and virulence genes in pathogenic Escherichia coli isolates from various sources

Shiga toxin-producing Escherichia coli (STEC) strains, including those of O157:H7 and the “big six” serogroups (i.e., O26, O45, O103, O111, O121, and O145) are food-borne pathogens that pose a serious health threat to humans. Ruminants, especially cattle, are a major reservoir for O157 and non-O157 STEC. In the present study, 115 E. coli strains isolated from small and very small beef processing plants were screened for virulence genes (stx1, stx2, eae) using a multiplex polymerase chain reaction (PCR). Thirteen (11.3%) of the 115 isolates tested positive for stx1, stx2, or eae genes, but only 4 (3.5%) tested positive for either stx1 or stx2. A multiplex PCR reaction targeting eight O-serogroups (O26, O45, O103, O111, O113, O121, O145, O157) identified 12 isolates as O26, O103, O111, or O145, with E. coli O26 being the most predominant serogroup (61.5%). The thirteen isolates were further analyzed using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) subtyping. Consistent with previous studies, CRISPR alleles from strains of the same serogroup were similar in their spacer content and order, regardless of the isolation source. A completely different CRISPR allele was observed in one isolate (“7-J”) which exhibited a different O-serogroup (O78). Our results confirmed previous findings that CRISPR loci are conserved among phylogenetically-related strains. In addition, 8 E. coli O26 isolates and a collection of 42 E. coli O26 isolates were screened for 12 enterohemorrhagic E. coli-specific genes. Seven genes (ECs848—Hypothetical Protein, ECs2226—Hypothetical Protein, ECs3857—nleB, ECs3858—Hypothetical Protein, ECs4552—escF, ECs4553—Hypothetical Protein, and ECs4557—sepL) were found in all 50 isolates. An additional 5 genes (ECs1322—ureA urease subunit γ, ECs1323—ureB urease subunit β, ECs1326—ureF, ECs1561—Hypothetical Protein, and ECs1568—Hypothetical Protein) were found to be highly prevalent in isolates from human sources, while lower in isolates from beef processing plants, cattle, and other sources. This finding indicates the possible role of these genes in virulence of human O26 strains.

Reduced Relapse Risk in Acute Myelogenous Leukemia (AML) after Allogeneic Hematopoietic Stem Cell Transplantation (HCT) in Patients Who Experienced CMV Reactivation: Influence of Donor KIR Genotypes

Background: Recent studies demonstrate an intriguing association between CMV reactivation and reduced risk of post-transplant AML relapse, yet mechanistic explanations remain elusive. Donor killer immunoglobulin-like receptor (KIR) genotypes have been associated with both AML and CMV clinical outcomes post allogeneic HCT. In this study we evaluated the CMV reactivation association with AML relapse risk, and the possible impact of donor KIR genotypes on the CMV-AML relapse relationship.Patients/Methods: A consecutive case-series of 288 patients with AML who underwent allogeneic HCT between 1995 and 2011 at City of Hope were identified, patients were excluded who were not in remission at HCT, or received ex-vivo/in-vivo T cell depletion. A landmark analysis on 276 patients who survived without relapse for at least 55 days (this time point was selected given that the majority of CMV reactivations had occurred by this time, with very few relapse events). The median age was 49 years (range: 18 - 70). All patients were in 1st (n=229) or 2nd remission (n=47). Transplant conditioning included fully ablative (n=151) or reduced intensity regimens (fludarabine 125mg/m2 + melphalan 140mg/m2: n=125). Patients received peripheral blood hematopoietic stem cell (n=240) or bone marrow (n=36) grafts from matched sibling donors (n=190) or unrelated donors (8/8 match: n=86). Graft-versus-host disease prophylaxis consisted of a calcineurin inhibitor plus sirolimus or a short course of methotrexate. No patients in this cohort received prophylactic anti-CMV therapy. CMV was monitored by shell vial culture or polymerase chain reaction (PCR) once or twice a week from day +21 to day +100. Preemptive therapy with ganciclovir or foscarnet began when CMV reactivation was detected by positive culture or PCR. KIR genotyping was performed on donor DNA samples as previously described (Tissue Antigens, 2004;64:462) using 28 primers in 4 multiplex PCR reactions for detection of 14 functional KIR genes.Results: CMV reactivation was observed in 85 of 276 patients (31%). The 5-year cumulative incidence of relapse was 30.2% (95% CI: 24.1 – 37.7%) in patients without CMV reactivation, significantly higher than in patients experiencing CMV reactivation (12.1%: 95% CI 6.7 – 21.6%, p=0.002) (Figure 1). Other clinical variables showing a significant association or p<0.10 by univariate analysis were: cytogenetic risk (high risk vs others: 37.6% vs. 20.0%, p= 0.01), transplant era (before or after 2005: 18.6% vs 28.0%, p=0.065), and conditioning (flu-mel vs others: 30.0% vs. 20.3%, p=0.095). In multivariate analysis, only CMV reactivation and high-risk cytogenetics were highly significant (Table 1). The association between CMV reactivation and reduced AML relapse was independent of the donor/recipient CMV serostatus.We then examined the protective effect of CMV reactivation on relapse in subgroups of patients based on KIR genotypes (available in 251 cases). A significant protective effect on 5-yr relapse incidence (RI) was observed in the group with donor KIR B/X genotype (>1 activating KIR gene, n=164): 11.7% RI (95% CI 5.5 – 24.8) in patients with CMV reactivation vs. 29.5% (95% CI 22.0 – 39.6) in those without (p=0.016). No significant differences (p=0.3) in relapse were seen in the A/A genotype (only 1 activating KIR gene, n=87). Likewise, when we evaluated an activating KIR gene, 2DS1, which is reported to protect from AML relapse, the significant association was preserved in donors with 2DS1 (n=96): 11.5% RI (95% CI 4.0 – 33.4) in patients with CMV reactivation vs. 30.7% (95% CI 21.5 – 43.9) in those without (p=0.05). No significant relationship (p=0.12) between relapse and CMV reactivation was seen in donors without 2DS1 (n=155).Conclusions: Our data confirm the earlier observation of a significant association between CMV reactivation and protection from AML relapse, and suggest that in patients with donor-derived activating KIRs, HLA-KIR interactions may augment the protection from AML relapse after CMV reactivation. Table 1Landmark Analysis of Cumulative Incidence of Relapse Event = Relapse, Completing risk = non-relapse mortalityVariablesValue# of eventsHazard Ratio(95% CI)p-valueCMV ReactivationNo (n=191) Yes* (n=85)n = 56 n = 112.6 (1.4 - 4.9)0.003Cytogenetic RiskFav/Int/Unknown*(n=207) Unfavorable (n=69)n = 42 n = 251.8 (1.1 - 2.9)0.018* reference group, Fav - favorable, Int - intermediate [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Development of Molecular Tools for Distinguishing Between the Highly Similar Rx1 and Rx2 PVX Extreme Resistance Genes in Tetraploid Potato

The Rx1 and Rx2 are extreme resistance genes, which have been introgressed from different species into potato cultivars and breeding lines. These two genes have a 98% and 96% sequence similarity at the nucleotide as well as at the amino acid level, respectively. Except one extra amino acid in the Rx2 gene, the high variations of the amino acid chain are due to single and double nucleotide variations, which are scattered throughout the coding regions. The high level of sequence similarity makes it complicated to identify these genes and to distinguish them from other highly similar genes, like the Gpa2 or from paralogous sequences by a single polymerase chain reaction (PCR). Here, we report the development of markers for the simple and rapid identification of the Rx1 as well as the Rx2 gene. Further, a multiplex PCR reaction is recommended for the simultaneous detection of both genes in a single reaction. Since these genes reside on different chromosomes, following their inheritance by the multiplex PCR method could help the easy incorporation of both genes into breeding lines. The detection method shown here could be routinely used in marker-assisted selection for Potato virus X extreme resistance and could enhance the effectiveness of potato breeding programs. Besides potato breeding, this method could also be effectively applied to mapping experiments as well as in research studies of resistance.

Simultaneous detection of waterborne viruses by multiplex real-time PCR

Norovirus, Rotavirus group A, the Hepatitis A virus, and Coxsackievirus are all common causes of gastroenteritis. Conventional diagnoses of these causative agents are based on antigen detection and electron microscopy. To improve the diagnostic potential for viral gastroenteritis, internally controlled multiplex real-time polymerase chain reaction (PCR) methods have been recently developed. In this study, individual real-time PCRs were developed and optimized for specific detections of Norovirus genogroup I, Norovirus genogroup II, Rotavirus group A, the Hepatitis A virus, and Coxsackievirus group B1. Subsequently, individual PCRs were combined with multiplex PCR reactions. In general, multiplex real-time PCR assays showed comparable sensitivities and specificities with individual assays. A retrospective clinical evaluation showed increased pathogen detection in 29% of samples using conventional PCR methods. Prospective clinical evaluations were detected in 123 of the 227 (54%) total samples used in the multiplex real-time PCR analysis. The Norovirus genogroup II was found most frequently (23%), followed by Rotavirus (20%), the Hepatitis A virus (4.5%), Coxsackievirus (3.5%), and Norovirus genogroup I (2.6%). Internally controlled multiplex real-time PCR assays for the simultaneous detection of Rotavirus, Coxsackievirus group B, the Hepatitis A virus, and Norovirus genogroups I and II showed significant improvement in the diagnosis of viral gastroenteritis.

Superantigen gene complement of Streptococcus pyogenes—relationship with other typing methods and short-term stability

The profiling of the superantigen (SAg) encoding genes has been frequently used as a complementary typing method for group A streptococci (GAS), but a confusing gene nomenclature and a large diversity of primers used in screening has led to some conflicting results. The aim of this work was to develop a polymerase chain reaction (PCR) method capable of efficiently amplifying all the known allelic variants of these genes, and to evaluate the congruence of this methodology with other commonly used molecular typing methods. The presence of the 11 known SAg genes and two other exotoxin-encoding genes (speB and speF) was tested in a collection of 480 clinical GAS isolates, using two multiplex PCR reactions. The SAg gene profile was compared with other typing methods. Four naturally occurring deletions involving the genes speB, speF, and rgg were characterized, two of which were found among invasive isolates. The absence of the chromosomally encoded genes speG and smeZ was supported by Southern blot hybridization and associated with specific GAS lineages, while the presence of phage-encoded genes was more variable. Positive associations between SAg genes or between SAg profiles and emm types or pulsed-field gel electrophoresis (PFGE) clusters were observed. The results suggest that the SAg profile diversifies faster than other properties commonly used for molecular typing, such as emm type and multilocus sequence typing (MLST) sequence types (STs), and can be a useful complement in GAS molecular epidemiology. Still, the short-term stability of the SAg gene profile among prevalent genetic lineages may largely explain the observed associations between SAg genes.

Development of a multiplex polymerase chain reaction assay for diarrheagenic Escherichia coli and Shigella spp. and its evaluation on colonies, culture broths, and stool

Detection of diarrheagenic Escherichia coli (DEC) typically depends on identification of virulence genes from stool cultures, not on stool itself. We developed a multiplex polymerase chain reaction (PCR) assay that detects key DEC virulence genes (stx1, stx2, eae, bfpA, ipaH, LT, STh, aaiC, aatA). The assay involved a multiplex PCR reaction followed by detection of amplicon(s) using Luminex beads. The assay was evaluated on over 100 colony and broth specimens. We then evaluated the assay using DNA extracted from stool, colony pools, and Gram-negative broths, using stool spiked with known quantities of DEC. Performance of the assay on stool DNA was most quantitative, while stool broth DNA offered the lowest limit of detection. The assay was prospectively evaluated on clinical specimens in Tanzania. Stool DNA yielded higher sensitivity than colony pools compared with broth DNA as the standard. We propose using this assay to screen for DEC directly in stool or stool broths.

Direct colony polymerase chain reaction (PCR): An efficient technique to rapidly identify and distinguish Mycosphaerella fijiensis and Mycosphaerella musicola

Sigatoka disease is the most important threat for banana production worldwide. Many species of Mycosphaerella have been described from banana but, to date, the three species Mycosphaerella fijiensis , M. musicola and M. eumusae are the only species found to be pathogenic to banana. Reliable identification by classical methods requires expertise because these fungi produce similar symptoms and they are morphologically similar. For studies of ecology, genetic diversity and epidemiology their differentiation is crucial. Several laboratories have developed molecular protocols to differentiate these fungi. Currently, a number of primers targeting ribosomal sequences, actin, tubulin and histone 3 genes are available for diagnosis of the Sigatoka complex. In the present work, we report a direct colony-polymerase chain reaction (DC-PCR) approach to rapidly distinguish M. fijiensis and M. musicola strains in multiplex PCR reactions. This is the most economical and the fastest procedure reported so far for diagnosis of these two Mycosphaerella species, which are distributed in banana-growing regions in the world; the DC-PCR technique was also found to be amenable for the identification of mating type of M. fijiensis isolates. This DC-PCR may also be applicable to prepare DNA templates for basic PCR-based analyses in other fungi. Keywords: Sigatoka diseases, Banana’s Mycosphaerella , molecular diagnosis, direct colony PCR

Comparison of molecular tests for the diagnosis of malaria in Honduras

BackgroundHonduras is a tropical country with more than 70% of its population living at risk of being infected with either Plasmodium vivax or Plasmodium falciparum. Laboratory diagnosis is a very important factor for adequate treatment and management of malaria. In Honduras, malaria is diagnosed by both, microscopy and rapid diagnostic tests and to date, no molecular methods have been implemented for routine diagnosis. However, since mixed infections, and asymptomatic and low-parasitaemic cases are difficult to detect by light microscopy alone, identifying appropriate molecular tools for diagnostic applications in Honduras deserves further study. The present study investigated the utility of different molecular tests for the diagnosis of malaria in Honduras.MethodsA total of 138 blood samples collected as part of a clinical trial to assess the efficacy of chloroquine were used: 69 microscopically confirmed P. falciparum positive samples obtained on the day of enrolment and 69 follow-up samples obtained 28 days after chloroquine treatment and shown to be malaria negative by microscopy. Sensitivity and specificity of microscopy was compared to an 18 s ribosomal RNA gene-based nested PCR, two single-PCR reactions designed to detect Plasmodium falciparum infections, one single-PCR to detect Plasmodium vivax infections, and one multiplex one-step PCR reaction to detect both parasite species.ResultsOf the 69 microscopically positive P. falciparum samples, 68 were confirmed to be P. falciparum-positive by two of the molecular tests used. The one sample not detected as P. falciparum by any of the molecular tests was shown to be P. vivax-positive by a reference molecular test indicating a misdiagnosis by microscopy. The reference molecular test detected five cases of P. vivax/P. falciparum mixed infections, which were not recognized by microscopy as mixed infections. Only two of these mixed infections were recognized by a multiplex test while a P. vivax-specific polymerase chain reaction (PCR) detected three of them. In addition, one of the day 28 samples, previously determined to be malaria negative by microscopy, was shown to be P. vivax-positive by three of the molecular tests specific for this parasite.ConclusionsMolecular tests are valuable tools for the confirmation of Plasmodium species and in detecting mixed infections in malaria endemic regions.

High Throughput Multiplex PCR and Probe-based Detection with Luminex Beads for Seven Intestinal Parasites

Polymerase chain reaction (PCR) assays for intestinal parasites are increasingly being used on fecal DNA samples for enhanced specificity and sensitivity of detection. Comparison of these tests against microscopy and copro-antigen detection has been favorable, and substitution of PCR-based assays for the ova and parasite stool examination is a foreseeable goal for the near future. One challenge is the diverse list of protozoan and helminth parasites. Several existing real-time PCR assays for the major intestinal parasites-Cryptosporidium spp., Giardia intestinalis, Entamoeba histolytica, Ancylostoma duodenale, Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis-were adapted into a high throughput protocol. The assay involves two multiplex PCR reactions, one with specific primers for the protozoa and one with specific primers for the helminths, after which PCR products are hybridized to beads linked to internal oligonucleotide probes and detected on a Luminex platform. When compared with the parent multiplex real-time PCR assays, this multiplex PCR-bead assay afforded between 83% and 100% sensitivity and specificity on a total of 319 clinical specimens. In conclusion, this multiplex PCR-bead protocol provides a sensitive diagnostic screen for a large panel of intestinal parasites.

New real-time PCR-based method for the joint genotyping of JAK2 (V617F) with inherited thrombophilic F5 and F2 mutations

Background During the last years the appearance of the acquired V617F mutation of the Janus Kinase 2 gene (JAK2) in patients suffering different thrombotic events has been described. We decided to develop a new and rapid multiplex real-time Polymerase Chain Reaction (PCR) in order to detect the V617F mutation together with the inherited prothrombotic mutations of factors F5 and F2. Design and methods The method was carried out on the LightCycler 2.0 (Roche Diagnostics, Mannheim, Germany) and consisted in a first step of simultaneous amplification by real-time PCR of the three genes to be genotyped, in a 20 μl closed tube, using a primer pair together with the correspondent FRET-hybridization probes for each gene. Results We assayed 41 samples in the multiplex PCR reaction, 19 were positive (46.34%) for V617F mutation. From the V617F positive samples we found 1 sample heterozygous for F2 (5.26%) and 1 sample heterozygous for F5 (5.26%), so a 10.52% of the samples tested combine V617F mutation with inherited thrombophilic mutations. Results were clear, rapid and reliable allowing a significant time saving. Conclusions The technique presented in this manuscript is a new achievement in the field of the molecular diagnosis that combines the genotyping of F5 and F2 with the assessment of the JAK2 (V617F) mutation load.