Multiplex PCR Protocol Research Articles

Detection and differentiation of Vibrio parahaemolyticus by multiplexed real-time PCR.

Vibrio parahaemolyticus is a common and important pathogen that causes human gastroenteritis worldwide. A rapid, sensitive, and specific assay is urgently required for detection and differentiation of V. parahaemolyticus strains. We designed three sets of primers and probes using groEL and two virulence genes (tdh and trh) from V. parahaemolyticus, and developed a multiplex real-time PCR protocol. The sensitivity and specificity of the multiplex assay was evaluated by environmental and clinical specimens of V. parahaemolyticus. The multiplex PCR response system and annealing temperature were optimized. The detection limits of the multiplex real-time PCR were 104 and 105 CFU/mL (or CFU/g) in pure cultures and spiked oysters, respectively. The multiplex real-time PCR specifically detected and differentiated V. parahaemolyticus from 35 Vibrio strains and 11 other bacterial strains. Moreover, this method can detect and distinguish virulent from nonvirulent strains, with no cross-reactivity observed in the bacteria tested. This newly established multiplex real-time PCR assay offers rapid, specific, and reliable detection of the total and pathogenic V. parahaemolyticus strains, which is very useful during outbreaks and sporadic cases caused by V. parahaemolyticus infection.

Yüksek oleik tip ayçiçeğinin güvenilir moleküler seçiminde yeni tasarlanmış primerler ile optimize PCR protokolü

High oleic sunflower is one of the most significant oilseed crops due to the stability of its oil in processing and desirable characteristics for health. Determination of high oleic sunflower by standard methods such as gas chromatography is time consuming and expensive. On the other hand, marker-assisted selection analysis with molecular markers associated with high oleic acid trait is a useful and powerful tool in order to facilitate sunflower breeding programs. In this study, we compared three molecular markers which have been used for selection of the high oleic sunflower varieties. We also describe an optimized PCR protocol with newly designed two primer pairs targeting normal sequence of FAD2 gene as internal control and direct analysis of the inserted DNA sequences which is known to be closely linked to the Pervenets mutation. According to results of our study, showing the insertion site which is linked to the Pervenets mutation by the insertion specific PCR protocols is more reliable than the SSR marker for selection of the high oleic sunflower varieties. Because we have not been able to get successful results with the available PCR protocols described for the insertion site, we report here a novel multiplex PCR protocol with newly designed primers enabling reliable discrimination of the high oleic and low oleic sunflower genotypes in a single PCR tube, which offers some advantages to the breeders by means of saving money and time.

A new multiplex PCR protocol to detect mixed trypanosomatid infections in species of Apis and Bombus.

Trypanosomatids are highly prevalent pathogens of Hymenoptera; however, most molecular methods used to detect them in Apis and Bombus spp. do not allow the identification of the infecting species, which then becomes expensive and time consuming. To overcome this drawback, we developed a multiplex PCR protocol to readily identify in a single reaction the main trypanosomatids present in these hymenopterans (Lotmaria passim, Crithidia mellificae and Crithidia bombi), which will facilitate the study of their epidemiology and transmission dynamics. A battery of primers, designed to simultaneously amplify fragments of the RNA polymerase II large subunit (RPB1) of L. passim, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of C. mellificae and the DNA topoisomerase II (TOPII) of C. bombi, was tested for target specificity under single and mixed template conditions using DNA extracted from cell cultures (L. passim ATCC PRA403; C. mellificae ATCC 30254) and from a bumblebee specimen infected with C. bombi only (14_349). Once validated, the performance of the method was assessed using DNA extractions from seven Apis mellifera (Linnaeus, 1758) and five Bombus terrestris (Linnaeus, 1758) field samples infected with trypanosomatids whose identity had been previously determined by PCR-cloning and sequencing (P-C-S). The new method confirmed the results obtained by P-C-S: two of the honeybee samples were parasitized by L. passim, C. mellificae and C. bombi at the same time, whereas the other five were infected with L. passim only. The method confirmed the simultaneous presence of L. passim and C. mellificae in two B. terrestris, where these parasites had not previously been reported.

Borehole water: a potential health risk to rural communities in South Africa

Abstract Sporadic outbreaks of diarrhoea in children in the Vhembe rural areas could be an indication of contamination in drinking water sources. In areas where improved water sources are used, not all rural households experience the benefits of these improved water sources. Water samples were collected from boreholes in three wards in the Vhembe District to determine microbiological risks over a 5-month period. A Water Point Mapping tool was used to indicate the borehole distribution. Water samples were taken from each functional borehole and analysed for total coliform and Escherichia coli counts, electrical conductivity, pH and temperature. A multiplex PCR protocol was used for identification of pathogenic E. coli. A total of 125 boreholes were identified of which only 12 were functional. Seven boreholes tested positive for total coliforms and E. coli counts. Four boreholes (33.3%) tested positive for diarrhoeagenic E. coli. Fifty-eight percent (58%) of water samples were without health risks, 17% were low risk and 25% could cause infection according to the South African water quality standards. This study indicated the importance of the role of the Municipalities and the maintenance plans that need to ensure that all boreholes are functional and provide safe drinking water to the rural communities.

Novel multiplex PCR reveals multiple trypanosomatid species infecting North American bumble bees (Hymenoptera: Apidae: Bombus).

Crithidia bombi and Crithidia expoeki (Trypanosomatidae) are common parasites of bumble bees (Bombus spp.). Crithidia bombi was described in the 1980s, and C. expoeki was recently discovered using molecular tools. Both species have cosmopolitan distributions among their bumble bee hosts, but there have been few bumble bee studies that have identified infections to species since the original description of C. expoeki in 2010. Morphological identification of species is difficult due to variability within each stage of their complex lifecycles, although they can be easily differentiated through DNA sequencing. However, DNA sequencing can be expensive, particularly with many samples to diagnose. In order to reliably and inexpensively distinguish Crithidia species for a large-scale survey, we developed a multiplex PCR protocol using species-specific primers with a universal trypanosomatid primer set to detect unexpected relatives. We applied this method to 356 trypanosomatid-positive bumble bees from North America as a first-look at the distribution and host range of each parasite in the region. Crithidia bombi was more common (90.2%) than C. expoeki (21.3%), with most C. expoeki-positive samples existing as co-infections with C. bombi (13.8%). This two-step detection method also revealed that 2.2% samples were positive for trypanosmatids that were neither C. bombi nor C. expoeki. Sequencing revealed that two individuals were positive for C. mellificae, one for Lotmaria passim, and three for two unclassified trypanosomatids. This two-step method is effective in diagnosing known bumble bee infecting Crithidia species, and allowing for the discovery of unknown potential symbionts.

Abstract P3-04-04: Germline and somatic mutation status in tissues from BRCA1/2 carriers

Abstract Background – aim: In carriers of BRCA1/2 pathogenic mutations (mut), it is expected that the germline mut is present in all tissues, particularly in normal; the somatic mut status in normal tissues from these patients is usually not addressed. We investigated the mut status in normal and tumor tissues in a real-life cohort of BRCA1/2 carriers who underwent prophylactic surgery. Methods: All 53 women had known BRCA1/2 germline mut that had been assessed independently; 42 had previous cancer manifestation (PCM); all had prophylactic mastectomy; 22 had prophylactic hystero-salpingo-oophorectomy. By using a 60-gene NGS panel, we examined the mut status of 231 samples, 39 peripheral blood and 192 paraffin tissues (FFPE: 46 tumors, out of which 43 breast; 97 normal breast [NB]; 49 normal ovary and salpinx [NGYN]). Germline mut status was interrogated in tissues with the above panel, Sanger sequencing and a multiplex PCR protocol for large exonic deletions, along with extensive FFPE DNA quality control (QC) to exclude false negatives. Results: Eight patients carried germline BRCA2 and 45 BRCA1 mut (29 in the BRCT-domain; 31 substitutions/indels). We identified somatic mut in 85% of the tumors and in 64% of the normal samples; mut were found significantly more often (p=0.003) and in higher numbers (p<0.001) in NGYN than in NB. In NB and NGYN, top 3 genes with somatic mut were BRCA2 (28%), BRCA1 (17%), TP53 (7%). In tumors, somatic mut were most frequent in TP53 (49%; p<0.001) and BRCA1 (38%; p=0.039). Among all tissue types, the 5 tumors post-neoadjuvant treatment had the highest and NB the lowest mut load (p=0.001). In NB and NGYN, mut load was not affected by PCM or BRCA1 mut domain but it was higher in BRCA1 vs. BRCA2 carriers (p=0.027) and in those with BRCA1 substitutions/indels vs. exon deleting and skipping mut (p<0.001). In tumors, germline BRCA1 substitutions/indels were associated with higher mut load (p=0.014). We validated germline mut status in all blood samples and in 111 tissue samples that passed FFPE DNA QC from 40 patients. The germline mut was not found in 14 samples (4 breast tumors; 3 NB; 7 NGYN) from 10 (25%) patients, all BRCA1 carriers, 9 with germline mut in the BRCT-domain. The only non-BRCT domain germline mut that was lost in one breast tumor, p.V1234fs, was replaced by the R1751* (validated), again in the BRCT domain. In normal tissues, those with lost germline mut had significantly less somatic mut compared to those with preserved germline mut (p<0.001). Conclusions: In BRCA1/2 carriers, somatic mut in BRCA genes and TP53 are present in normal breast and GYN tissues, more frequently in the latter, and seem associated with the mutated gene and with the type of mut in the germline. The mut status of normal breast tissue does not seem to be affected by neoadjuvant chemotherapy for breast cancer. The observed BRCA1 germline mut loss, particularly in normal tissues, may be approached as a negative selection for the inherited mut; similarly to the described germline mut reversion after chemotherapy, tissues may react to deleterious effects of haploinsufficiency, which needs functional validation. Citation Format: Kotoula V, Demiri E, Fostira F, Vrettou E, Papadopoulou K, Tikas I, Papazisis K, Zaramboukas T, Asimaki-Vlachopoulou A, Miliaras S, Fountzilas E, Ananiadis A, Chrisafi S, Poulios C, Natsiopoulos I, Tsiftsoglou A, Fountzilas G. Germline and somatic mutation status in tissues from BRCA1/2 carriers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-04-04.

An experimental study for rapid detection and quantification of endodontic microbiota following photo-activated disinfection via new multiplex real-time PCR assay.

The infected root canal system harbors one of the highest accumulations of polymicrobial infections. Since the eradication of endopathogenic microbiota is a major goal in endodontic infection therapy, photo-activated disinfection (PAD) can be used as an alternative therapeutic method in endodontic treatment. Compared to cultivation-based approaches, molecular techniques are more reliable for identifying microbial agents associated with endodontic infections. The purpose of this study was to evaluate the ability of designed multiplex real-time PCR protocol for the rapid detection and quantification of six common microorganisms involved in endodontic infection before and after the PAD. Samples were taken from the root canals of 50 patients with primary and secondary/persistent endodontic infections using sterile paper points. PAD with toluidine blue O (TBO) plus diode laser was performed on root canals. Resampling was then performed, and the samples were transferred to transport medium. Then, six target microorganisms were detected using multiplex real-time PCR before and after the PAD. Veillonella parvula was found using multiplex real-time PCR to have the highest frequency among samples collected before the PAD (29.4%), followed by Porphyromonas gingivalis (23.1%), Aggregatibacter actinomycetemcomitans (13.6%), Actinomyces naeslundii (13.0%), Enterococcus faecalis (11.5%), and Lactobacillus rhamnosus (9.4%). After TBO-mediated PAD, P. gingivalis strains, the most resistance microorganisms, were recovered in 41.7% of the samples using molecular approach (P > 0.05). As the results shown, multiplex real-time PCR as an accurate detection approach with high-throughput and TBO-mediated PAD as an efficient antimicrobial strategy due to the significant reduction of the endopathogenic count can be used for detection and treatment of microbiota involved in infected root canals, respectively.

Classification of Avian Pathogenic Escherichia coli (APEC) and Human Uropathogenic Escherichia coli (UPEC) in Phylogenetic Groups and Association with Pathogenicity In Vivo

Background: Avian pathogenic E. coli (APEC) and uropathogenic E. coli (UPEC) are responsible, respectively, for avian colibacillosis and for 80% of urinary tract infections in humans. E. coli control is difficult due to the absence of a reliable method to differentiate pathogenic and commensal strains. Genetic similarity between APEC and UPEC suggests a common ancestral origin and the capability of potentially pathogenic strains to affect human health. The classification in phylogenetic groups facilitates the identification of pathogenic strains. The objective of this work was to classify APEC and UPEC E. coli strains into phylogenetic groups and to associate it with in vivo pathogenicity.Materials, Methods & Results: 460 APEC and 450 UPEC strains, stored in BHI with glycerol at -80°C, were selected. APEC strains were isolated from cellulitis, respiratory tract and poultry litter of broiler flocks from Southern Brazil. The UPEC strains from urinary tract infection were provided by a hospital in Porto Alegre. After DNA extraction, APEC and UPEC strains were classified into four phylogenetic groups (A, B1, B2 and D) by a multiplex-PCR protocol for the detection of the chuA and yjaA genes and the TspE4.C2 DNA fragment. Phylogenetic groups were associated with pathogenicity indexes (PI), presented on a scale of 0 to 10, which were previously obtained through the inoculation of APEC strains in one-day-old chicks. Phylogenetic groups were also associated with the presence of 38 virulence-associated genes. The multiplex-PCR protocol was able to differentiate 100% of the APEC and UPEC strains in the four phylogenetic groups. The majority of APEC strains were classified into phylogenetic groups D (31.1%) and B2 (24.1%). On the other hand, the majority of UPEC strains were classified into B2 (53.6%). Among APEC strains, five genes (crl, mat, ompA, fimC and fimH) were detected in more than 80% of strains in all groups. Some genes showed a significant association with specific phylogenetic groups. Gene ireA was exclusively to group D, kpsMT II and cvaC to B2 and sat was exclusively to B1. Four genes (ireA, sfa/focCD, ibeA, tsh) were detected in more than 70% of UPEC strains in all phylogenetic groups. Gene iroN1 showed a significant association exclusively to group A, and iucD, papC and irp2 to B1 group. APEC isolated from poultry litter presented significantly lower PIs than those isolated from cellulitis and from birds with respiratory signs. The average PI from B2 group was significantly higher than that of D group. In addition, the PIs of the two groups were significantly higher than those of A and B1.Discussion: The high frequency of UPEC classified as B2 is in agreement with the literature. More virulent strains are usually classified into B2 group and some of them may be classified into D group. On the other hand, the distribution of APEC isolates in phylogenetic groups is characterized by variability and it is usually related to the origin of the isolates, as observed in the study. Since E. coli strains isolated from human and poultry face similar challenges in infection establishment of extraintestinal sites, they may share some virulence genes. In this study, most of the 38 genes presented a high frequency in both APEC and UPEC strains. As the distribution of APEC strains in phylogenetic groups showed a significant association with pathogenicity, multiplex-PCR becomes an important tool for screening the pathogenicity of strains isolated from the poultry production chain.

Diagnostic accuracy of the ROCHE Septifast PCR system for the rapid detection of blood pathogens in neonatal sepsis-A prospective clinical trial.

IntroductionDiagnosis of neonatal sepsis remains a major challenge in neonatology. Most molecular-based methods are not customized for neonatal requirements. The aim of the present study was to assess the diagnostic accuracy of a modified multiplex PCR protocol for the detection of neonatal sepsis using small blood volumes.Methods212 episodes of suspected neonatal late onset sepsis were analyzed prospectively using the Roche SeptiFast® MGRADE PCR with a modified DNA extraction protocol and software-handling tool. Results were compared to blood culture, laboratory biomarkers and clinical signs of sepsis.ResultsOf 212 episodes, 85 (40.1%) were categorized as “not infected”. Among these episodes, 1 was false positive by blood culture (1.2%) and 23 were false positive by PCR (27.1%). Of 51 (24.1%) episodes diagnosed as “culture proven sepsis”, the same pathogen was detected by blood culture and PCR in 39 episodes (76.5%). In 8 episodes, more pathogens were detected by PCR compared to blood culture, and in 4 episodes the pathogen detected by blood culture was not found by PCR. One of these episodes was caused by Bacillus cereus, a pathogen not included in the PCR panel. In 76/212 (35.8%) episodes, clinical sepsis was diagnosed. Among these, PCR yielded positive results in 39.5% of episodes (30/76 episodes). For culture-positive sepsis, PCR showed a sensitivity of 90.2% (95%CI 86.2–94.2%) and a specificity of 72.9% (95%CI 67.0–79.0%).ConclusionThe Roche SeptiFast® MGRADE PCR using a modified DNA extraction protocol showed acceptable results for rapid detection of neonatal sepsis in addition to conventional blood culture. The benefit of rapid pathogen detection has to be balanced against the considerable risk of contamination, loss of information on antibiotic sensitivity pattern and increased costs.

Heritability estimate for mantle edge pigmentation and correlation with shell pigmentation in the white-shell strain of Pacific oyster, Crassostrea gigas

The pacific oyster (Crassostrea gigas) is one of the most widely farmed aquatic animal species and of great economic importance. Mantle edge color in C. gigas is highly variable and considered as a new potential trait for a better commercial value. In this study, heritability for mantle edge pigmentation and its correlation with shell pigmentation were explored in the white-shell strain of C. gigas with mixed-family method. A total of 460 offspring raised under communal conditions were successfully assigned to their parents using four multiplex PCR protocols based on 11 microsatellite loci. Mantle edge pigmentation was measured through assay of melanin content using spectrophotometric analysis. Animal model heritability estimate were 0.215±0.092 for mantle edge pigmentation. The genetic and phenotypic correlations between two pigment traits were 0.980±0.094 and 0.423±0.042, respectively. Pearson analyses for average mantle edge and shell pigmentation of family also showed strong and significant correlation (r=0.729, P=0.000, n=29). Chi-square analyses showed that mantle edge pigmentation of family 7 segregated into “lighter” and “darker” groups in a 3:1 ratio (P=0.833), suggesting that a major locus might control mantle edge pigment trait. The results obtained in this study will benefit selective breeding of C. gigas with desired mantle and shell pigmentation.

An effective molecular approach for assessing cereal aphid-parasitoid-endosymbiont networks

Molecular approaches are increasingly being used to analyse host-parasitoid food webs as they overcome several hurdles inherent to conventional approaches. However, such studies have focused primarily on the detection and identification of aphids and their aphidiid primary parasitoids, largely ignoring primary parasitoid-hyperparasitoid interactions or limiting these to a few common species within a small geographical area. Furthermore, the detection of bacterial secondary endosymbionts has not been considered in such assays despite the fact that endosymbionts may alter aphid-parasitoid interactions, as they can confer protection against parasitoids. Here we present a novel two-step multiplex PCR (MP-PCR) protocol to assess cereal aphid-primary parasitoid-hyperparasitoid-endosymbiont interactions. The first step of the assay allows detection of parasitoid DNA at a general level (24 primary and 16 hyperparasitoid species) as well as the species-specific detection of endosymbionts (3 species) and cereal aphids (3 species). The second step of the MP-PCR assay targets seven primary and six hyperparasitoid species that commonly occur in Central Europe. Additional parasitoid species not covered by the second-step of the assay can be identified via sequencing 16S rRNA amplicons generated in the first step of the assay. The approach presented here provides an efficient, highly sensitive, and cost-effective (~consumable costs of 1.3 € per sample) tool for assessing cereal aphid-parasitoid-endosymbiont interactions.

Parentage analysis of tropical spiny lobster (Panulirus homarus) by microsatellite markers

Parentage analysis is of vital importance for hatchery production and breeding programmes. Two multiplex PCR protocols including seven tropical spiny lobster (Panulirus homarus) microsatellites (Pho-G06, Pho-G25, Pho-G53, Pho-G62, Pho-G74, Pho-G89 and Pho-G100) were introduced for parentage assignment. All loci were polymorphic with allele sizes from 113 to 337 base pairs (bp), observed alleles (k) from two to seven and polymorphic information content (PIC) of 0.25 to 0.73. Twenty-four stage-3 phyllosoma larvae (39 days posthatching) were collected for paternity exclusion study using selected microsatellites. Exclusion-based parentage analysis unambiguously assigned 83% of fry (20 of 24) to a single female parent. Of ten putative female parents, five have contributed to the 20 allocated offspring, with one being true parent of 11. Four others were assigned to two or more potential female parents, probably due to genotyping error or the presence of null allele. The exclusion power (EP) for the seven loci varied between 13% and 54% with known genotypes of one parent (P1) and 19% and 71% for given the genotypes of both parents (P2). The theoretical combined parentage exclusion (cEP) power for all seven microsatellites was P1 = 95% and P2 = 99%. The power of discrimination for each locus varied between 0.18 and 0.86. This report presents the first study to utilize microsatellite markers for successful parentage assignment of P. homarus. The selected microsatellites provide a practical tool for parentage analysis in hatchery production of juveniles as well as in future commercial breeding programme of tropical spiny lobsters.

Development of multiplex PCR for simultaneous detection and differentiation of six DNA and RNA viruses from clinical samples of sheep and goats

Multiplex reverse transcription-polymerase chain reaction (RT-PCR) and PCR protocols were developed and subsequently evaluated for its effectiveness in detecting simultaneously single and mixed infections in sheep and goats. Specific primers for three DNA viruses and three RNA viruses, including foot and mouth disease virus (FMDV), Bluetongue virus (BTV), peste des petits ruminants virus (PPRV), sheeppox virus (SPPV), goatpox virus (GTPV) and orf virus (ORFV) were used for testing procedure. A single nucleic acid extraction protocol was adopted for the simultaneous extraction of both RNA and DNA viruses. The multiplex PCR consisted with two-step procedure which included reverse transcription of RNA virus and multiplex PCR of viral cDNA and DNA. The multiplex PCR assay was shown to be sensitive because it could detect at least 100pg of viral genomic DNA or RNA from a mixture of six viruses in a reaction. The assay was also highly specific in detecting one or more of the same viruses in various combinations in specimens. Thirty seven clinical samples collected from sheep and goats were detected among forty three samples tested by both uniplex and multiplex PCR, showing highly identification. As results of the sensitivity and specificity, the multiplex PCR is a useful approach for clinical diagnosis of mixed infections of DNA and RNA viruses in sheep and goats with a reaction.

Multiplex PCR-based identification of Streptococcus canis, Streptococcus zooepidemicus and Streptococcus dysgalactiae subspecies from dogs.

Streptococcus canis (S. canis), Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) and Streptococcus dysgalactiae subspecies (S. dysgalactiae subspecies) are β-haemolytic Gram positive bacteria infecting animals and humans. S. canis and S. zooepidemicus are considered as two of the major zoonotic species of Streptococcus, while more research is needed on S. dysgalactiae subspecies bacteria. In this work, a multiplex-PCR protocol was tested on strains and clinical samples to detect S. canis, S. dysgalactiae subspecies and S. equi subspecies bacteria in dogs. All strains were correctly identified as S. canis, S. equi subspecies or S. dysgalactiae subspecies by the multiplex-PCR. The main Streptococcus species isolated from symptomatic dogs were confirmed S. canis. The multiplex-PCR protocol described is a rapid, accurate and efficient method for identifying S. canis, S. equi subspecies and S. dysgalactiae subspecies in dogs and could be used for diagnostic purposes and for epidemiological studies.

Detection of Emerging Food Pathogens in Chicken Meat Using Multiplex Polymerase Chain Reaction

<p>A study was undertaken to develop a multiplex PCR (m-PCR) protocol for simultaneous detection of <em>Campylobacter jejuni </em>and <em>Listeria monocytogenes </em>in chicken meat. The extraction of DNA was carried out using commercial DNA extraction kit, Phenol Chloroform and boiling method. Samples with OD ratio (260:280) between 1.7 and 1.9 were considered good in terms of concentration and purity and were used for PCR amplification. DNA extraction kit and Phenol Chloroform method revealed good OD value were used for sample extraction process. PCR and m-PCR amplification was carried out using genus specific primers were designed by targeting its<em> Hyp </em>(500 bp) and <em>prfA </em>(290 bp) gene for <em>Campylobacter jejuni </em>and <em>Listeria monocytogenes</em> respectively. Electrophoresis of amplified PCR products and gel documentation revealed 500 bp and 290 bp in 2% Agarose.<strong> </strong>The multiplex PCR technique was standardized using the reference strains with the similar amplification procedure. The minimum detection level (sensitivity) by mPCR for <em>Campylobacter jejuni </em>and <em>Listeria monocytogenes</em> was found to be 0.2 ng/ul and 1.0 ng/ul of DNA in a reaction mixture (25 ul). The developed multiplex PCR technique could detect <em>Campylobacter jejuni </em>and <em>Listeria monocytogenes</em> upto 3 × 10<sup>5</sup> and 3 × 10<sup>4</sup> CFU/ml of artificially inoculated meat homogenate. Around 60 chicken meat samples were collected from different regions of chennai and were screened for the presence of <em>Campylobacter jejuni</em> and <em>Listeria monocytogenes</em>. All the samples screened were not positive either for <em>Campylobacter jejuni</em> and <em>Listeria monocytogenes</em>. The negative samples were further checked by culture methods and good correlation between these two methods was observed. Hence, the m-PCR technique developed in this study can be used as a rapid screening test for detection of <em>Campylobacter jejuni </em>and <em>Listeria monocytogenes</em> from chicken meat within 24 hours.</p>

MAT gene idiomorphs suggest a heterothallic sexual cycle in the citrus pathogen Phyllosticta citricarpa

Sexual reproduction in fungi is controlled by mating type genes, which are located at the MAT locus. In this study, we investigated the structure of this locus in the phytopathogenic fungus Phyllosticta citricarpa, the causal agent of citrus black spot disease. Despite intensive study, its sexual state has never been observed in single-spore culture. Through analysis of the genome sequences of two individual P. citricarpa isolates, the sequence of the DNA lyase gene was identified and, as previously reported in the literature, the mating type genes were located in the 3′ flanking region of this gene. The results suggested that P. citricarpa is heterothallic, owing to the exclusive presence of the MAT1–1 or MAT1–2 gene in individual strains. In order to characterize the MAT locus, we designed primers to amplify this region. P. citricarpa was found to have complete and apparently functional copies of MAT genes, containing α-1 and HMG domains, present in different isolates. In addition to MAT1–2-1 and MAT1–1-1 genes, the MAT1–1-4 gene was located in the 5′ flanking region of the MAT1–1-1 gene and the MAT1–2-5 gene was located in 5′ flanking region of the MAT1–2-1 gene. A multiplex PCR protocol was also developed to differentiate P. citricarpa idiomorphs, which can be used in distribution and incidence studies of mating type strains, in order to determine the occurrence of sexual reproduction and to facility crossing studies. Furthermore, in Brazil, the two idiomorphs occur in a 1:1 ratio, which is expected in sexually reproducing populations.

First record of wild boar infected with Trichinella pseudospiralis in Poland

Abstract Introduction: The paper describes identification of Trichinella species isolated from wild boars (Sus scrofa) in the most popular hunting region of the West Pomeranian Province of Poland. Material and Methods: The Trichinella larvae were identified by digestion method. For species identification of the larvae, multiplex PCR was used according to the European Reference Laboratory for Parasites Multiplex PCR protocol. The results were confirmed by molecular amplification of 5S rDNA gene and sequence analysis. Results: Prevalence of 0.54% Trichinella–positive wild boars in the West Pomeranian Province was recorded. Examination of the larvae showed the occurrence of T. spiralis in 79 %, T. britovi in 16.5 %, mixed infection with T. spiralis/T. britovi in 3.5%, and T. pseudospiralis in 1.0% of the boars. Conclusion: This is the first record of wild boar infected with non-encapsulated larvae of T. pseudospiralis in Poland. The species is very difficult to determine, especially using trichinoscopic method. The discovery of the larvae in the animals which may be intended for human consumption confirms that digestion technique should be the only method used for the inspection of meat, especially that from wild boars..

Implementation and Evaluation of a Fully Automated Multiplex Real-Time PCR Assay on the BD Max Platform to Detect and Differentiate Herpesviridae from Cerebrospinal Fluids.

A fully automated multiplex real-time PCR assay—including a sample process control and a plasmid based positive control—for the detection and differentiation of herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2) and varicella-zoster virus (VZV) from cerebrospinal fluids (CSF) was developed on the BD Max platform. Performance was compared to an established accredited multiplex real time PCR protocol utilizing the easyMAG and the LightCycler 480/II, both very common devices in viral molecular diagnostics. For clinical validation, 123 CSF specimens and 40 reference samples from national interlaboratory comparisons were examined with both methods, resulting in 97.6% and 100% concordance for CSF and reference samples, respectively. Utilizing the BD Max platform revealed sensitivities of 173 (CI 95%, 88–258) copies/ml for HSV1, 171 (CI 95%, 148–194) copies/ml for HSV2 and 84 (CI 95%, 5–163) copies/ml for VZV. Cross reactivity could be excluded by checking 25 common viral, bacterial and fungal human pathogens. Workflow analyses displayed shorter test duration as well as remarkable fewer and easier preparation steps with the potential to reduce error rates occurring when manually assessing patient samples. This protocol allows for a fully automated PCR assay on the BD Max platform for the simultaneously detection of herpesviridae from CSF specimens. Singular or multiple infections due to HSV1, HSV2 and VZV can reliably be differentiated with good sensitivities. Control parameters are included within the assay, thereby rendering its suitability for current quality management requirements.

Alternaria alternata apple pathotype (A. mali) causes black spot of European pear

A disease caused by Alternaria alternata occurred on the leaves of European pear cultivar Le Lectier in Niigata Prefecture, Japan, and was named black spot of European pear. In conidial inoculation tests, the causal pathogen induced not only small black lesions on the leaves of European pear cultivar Le Lectier, but severe lesions on the leaves of apple cultivar Red Gold, which is susceptible to the A. alternata apple pathotype (previously called A. mali) causing Alternaria blotch of apple. Interestingly, the apple pathotype isolate showed the same pathogenicity as the European pear pathogen. HPLC analysis of the culture filtrates revealed that A. alternata causing black spot of European pear produced AM-toxin I, known as a host-specific toxin of the A. alternata apple pathotype. AM-toxin I induced veinal necrosis on leaves of Le Lectier and General Leclerc cultivars, both susceptible to the European pear pathogen, at 5 × 10−7 M and 10−6 M respectively, but did not affect leaves of resistant cultivars at 10−4 M. PCR analysis with primers that specifically amplify the AM-toxin synthetase gene detected the product of expected size in the pathogen. These results indicate that A. alternata causing black spot of European pear is identical to that causing Alternaria blotch of apple. This is the first report of European pear disease caused by the A. alternata apple pathotype. This study provides a multiplex PCR protocol, which could serve as a useful tool, for the epidemiological survey of these two diseases in European pear and apple orchards.

Identification and Molecular Analysis of Pasteurella Multocida Isolated from Rabbits

A total of 117 samples were collected from individual cases of diseased rabbits showed respiratory signs (68 samples from nasopharyngeal swabs, 43 samples from lung tissues, 2 samples from subcutaneous abscesses swabs, 3 samples from liver swabs and 1 swab from the conjunctiva). During this study 11 Pasteurella multocida isolates were isolated with percentage of 9.4% as 7 isolates from nasal swabs (6%) and 4 isolates from lung tissues (3.4%). The samples examined phenotypically (morphology and biochemical characters of Pasteurella multocida) and tested by PCR technique for confirmatory identification and detected the presence of capsule, hemagglutinin and type IV fimbria genes as virulence factors. This study confirmed the presence of capsule in 100% of the examined samples (6/6) and hemagglutinin in 83.3% (5/6) examined samples and type IV fimbria in 33.3% (2/6) examined samples. It was concluded that all the examined Pasteurella multocida samples in this study were virulent and the multiplex-PCR protocols could be useful tools for rapid and simultaneous detection of Pasteurella multocida virulence genes.