Specific Polymerase Chain Reaction Assay Research Articles

Development of three triplex real-time reverse transcription PCR assays for the qualitative molecular typing of the nine serotypes of African horse sickness virus

Blood samples collected as part of routine diagnostic investigations from South African horses with clinical signs suggestive of African horse sickness (AHS) were subjected to analysis with an AHS virus (AHSV) group specific reverse transcription quantitative polymerase chain reaction (AHSV RT-qPCR) assay and virus isolation (VI) with subsequent serotyping by plaque inhibition (PI) assays using AHSV serotype-specific antisera. Blood samples that tested positive by AHSV RT-qPCR were then selected for analysis using AHSV type specific RT-qPCR (AHSV TS RT-qPCR) assays. The TS RT-qPCR assays were evaluated using both historic stocks of the South African reference strains of each of the 9 AHSV serotypes, as well as recently derived stocks of these same viruses. Of the 503 horse blood samples tested, 156 were positive by both AHSV RT-qPCR and VI assays, whereas 135 samples that were VI negative were positive by AHSV RT-qPCR assay. The virus isolates made from the various blood samples included all 9 AHSV serotypes, and there was 100% agreement between the results of conventional serotyping of individual virus isolates by PI assay and AHSV TS RT-qPCR typing results. Results of the current study confirm that the AHSV TS RT-qPCR assays for the identification of individual AHSV serotypes are applicable and practicable and therefore are potentially highly useful and appropriate for virus typing in AHS outbreak situations in endemic or sporadic incursion areas, which can be crucial in determining appropriate and timely vaccination and control strategies.

Clostridium butyricum Strains and Dysbiosis Linked to Necrotizing Enterocolitis in Preterm Neonates.

Necrotizing enterocolitis (NEC) is the most common and serious gastrointestinal disorder among preterm neonates. We aimed to assess a specific gut microbiota profile associated with NEC. Stool samples and clinical data were collected from 4 geographically independent neonatal intensive care units, over a 48-month period. Thirty stool samples from preterm neonates with NEC (n = 15) and controls (n = 15) were analyzed by 16S ribosomal RNA pyrosequencing and culture-based methods. The results led us to develop a specific quantitative polymerase chain reaction (qPCR) assay for Clostridium butyricum, and we tested stool samples from preterm neonates with NEC (n = 93) and controls (n = 270). We sequenced the whole genome of 16 C. butyricum strains, analyzed their phylogenetic relatedness, tested their culture supernatants for cytotoxic activity, and searched for secreted toxins. Clostridium butyricum was specifically associated with NEC using molecular and culture-based methods (15/15 vs 2/15; P < .0001) or qPCR (odds ratio, 45.4 [95% confidence interval, 26.2-78.6]; P < .0001). Culture supernatants of C. butyricum strains from preterm neonates with NEC (n = 14) exhibited significant cytotoxic activity (P = .008), and we identified in all a homologue of the β-hemolysin toxin gene shared by Brachyspira hyodysenteriae, the etiologic agent of swine dysentery. The corresponding protein was secreted by a NEC-associated C. butyricum strain. NEC was associated with C. butyricum strains and dysbiosis with an oxidized, acid, and poorly diversified gut microbiota. Our findings highlight the plausible toxigenic mechanism involved in the pathogenesis of NEC.

Quantitative analysis of the mRNA expression levels of BCL2 and BAX genes in human osteoarthritis and normal articular cartilage: An investigation into their differential expression.

Osteoarthritis (OA) is primarily characterized by articular cartilage degeneration and chondrocyte loss. Although the role of apoptosis in cartilage pathobiology remains to be elucidated, the apoptotic B‑cell CLL/lymphoma 2 (BCL2) gene family is considered to be involved in OA. The purpose of the present study was to quantitatively analyze the mRNA expression profiles of the BCL2‑associated X protein (BAX) and BCL2 genes in human OA and in normal cartilage. Cartilage tissue samples were obtained from 78 patients undergoing total knee arthroplasty for OA (OA group) and orthopedic interventions for causes other than OA (control group). Total RNA was isolated from the cartilage tissue specimens and reverse transcribed into cDNA. A highly sensitive and specific reverse transcription quantitative polymerase chain reaction assay was developed for quantification of the mRNA levels of BAX and BCL2, using beta‑2 microglobulin as an endogenous control for normalization purposes. Gene expression analysis was performed using the comparative Ct (2(‑ΔΔCt)) method. The mRNA expression of BAX presented an increasing trend in the OA group compared with the control group, although without statistically significace (P=0.099). By contrast, the expression ratio of BCL2/BAX was found to be significantly decreased (2.76‑fold) in the OA group compared with the normal cartilage control group (P=0.022). A notable 4.6‑fold overexpression of median mRNA levels of BAX was also observed in patients with stage III OA compared with the control (P=0.034), while the BCL2/BAX ratio was markedly (2.5‑fold) decreased (P=0.024). A marked positive correlation was observed between the mRNA levels of BAX and BCL2 in the control group (r(s)=0.728; P<0.001), which was also present in the OA group, although to a lesser degree (r(s)=0.532; P<0.001). These results further implicate apoptosis in the pathogenesis of OA, through molecular mechanisms, which include the aberrant expression of the BCL2 gene family. Further investigation may reveal novel prognostic biomarkers and potential targets for therapeutic interventions in the early stages of OA.

Garlic (A. sativum L.) alliinase gene family polymorphism reflects bolting types and cysteine sulphoxides content.

BackgroundAlliinase is an important enzyme occurring in Allium species that converts precursors of sulfuric compounds, cysteine sulfoxides into a biologically active substance termed allicin. Allicin facilitates garlic defense against pests and produces health-promoting compounds. Alliinase is encoded by members of a multigene family that has not yet been sufficiently characterized, namely with regard to the copy numbers occurring within the genome and the polymorphisms among the family members.ResultsWe cloned 45 full-length alliinase amplicons of cultivar (cv.) Jovan. Sequence analyses revealed nine different sequence variants (SVs), confirming the multilocus nature of this gene family. Several mutations in exons, mainly occurring in the first exon coding for vacuolar signal peptide, were found. These results enabled us to identify sequences with putatively modified vacuole-targeting abilities. We found additional sequence variants using partial amplicons. We estimated that the minimum number of gene copies in the diploid genome of the investigated cultivar was fourteen. We obtained similar results for another three cultivars, which differed in bolting type and place of origin. The further identification of high degree of polymorphisms in the intron regions allowed us to develop a specific polymerase chain reaction assay capable to capture intron length polymorphism (ILP). This assay was used to screen 131 additional accessions. Polymorphic data were used for cluster analysis, which separated the bolting and non-bolting garlic types and those with high cysteine-sulfoxide contents in a similar way as AFLP analysis in previous study. These newly developed markers can be further applied for the selection of desirable garlic genotypes.ConclusionsDetailed analysis of sequences confirmed multigenic nature of garlic alliinase. Intron and exon polymorphism analysis generated similar results as whole genome variability assessed previously by AFLP. Detected polymorphism is thus also associated with cysteine-sulphoxide content in individual genotypes. ILP markers capable to detect intron polymorphisms were newly developed. Developed markers could be applied in garlic breeding. Higher genetic variability found in bolting genotypes may indicates longer period of their sexual propagation in comparison with nonbolting genotypes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-015-0214-z) contains supplementary material, which is available to authorized users.

PCR-Mediated Detection of Endophytic and Phytopathogenic Fungi from Needles of the Japanese Black Pine, &amp;lt;i&amp;gt;Pinus thunbergii&amp;lt;/i&amp;gt;

A specific and sensitive polymerase chain reaction (PCR) assay based on the internal transcribed spacer (ITS) region of rDNA sequences was developed to detect endophytic and phytopathogenic fungi from needles of the Japanese black pine, Pinus thunbergii. Sequences of the ITS regions of Lophodermium conigenum, Lecanosticta acicola, Pestalotiopsis neglecta, Rhizosphaera kalkhoffii, and Septorioides pini-thunbergii were compared, and each specific primer pair for these species was designed. First, the designed primer pairs were tested for their specificity to detect each species. A PCR product was amplified only each combination of species and its specific primer pair, confirming the specificity of the designed primer pairs. These primer pairs were also tested on DNA extracted from the needles of P. thunbergii. The PCR products were amplified not only in needles with lesions but also in healthy needles without symptoms. Furthermore, several endophytic and phytopathogenic fungi could be simultaneously detected from the same region in a needle. The PCR-mediated detection method developed in this study will be a valuable tool for the detection of the endophytic and phytopathogenic fungi, not only as a rapid diagnostic tool for early detection but also for monitoring variations in both the quality and quantity of the endophytic and phytopathogenic fungi in needles in Japanese black pines.

Isolation and genetic characterization of an Actinobacillus pleuropneumoniae serovar K12:O3 strain.

An atypical Actinobacillus pleuropneumoniae serovar 12 strain, termed QAS106, was isolated from a clinical case of porcine pleuropneumonia in Japan. An immunodiffusion (ID) test identified the strain as serovar 12. However, the ID test also demonstrated that strain QAS106 shared antigenic determinants with both the serovar 3 and 15 reference strains. Strain QAS106 was positive in the capsular serovar 12-specific polymerase chain reaction (PCR) assay, while the PCR toxin gene profiling and omlA PCR typing assays indicated that strain QAS106 was similar to serovar 3. The nucleotide sequence of the 16S ribosomal DNA (rDNA) of strain QAS106 was identical with that of serovars 3 and 12, but it showed 99.7% identity with that of serovar 15. Nucleotide sequence analysis revealed that genes involved in biosynthesis of the capsular polysaccharide (CPS) of strain QAS106 were identical to those of serovar 12 at the amino acid level. On the other hand, strain QAS106 would express putative proteins involved in the biosynthesis of lipopolysaccharide (LPS) O-polysaccharide (O-PS), the amino acid sequences of which were identical or nearly identical to those of serovars 3 and 15. In conclusion, strain QAS106 should be recognized as K12:O3, even though typical serovar 12 strains are K12:O12. The emergence of an atypical A. pleuropneumoniae serovar 12 strain expressing a rare combination of CPS and O-PS antigens would hamper precise serodiagnosis by the use of either CPS- or LPS-based serodiagnostic methodology alone.

Resistance to Clover yellow vein virus in Common Bean Germplasm

ABSTRACTClover yellow vein virus (ClYVV) is a potentially devastating viral pathogen of temperate common bean (Phaseolus vulgaris L.) production. Recent research demonstrated that resistance to ClYVV is conditioned by bc‐3 and bc‐32 and that a nonsynonymous mutation in P. vulgaris eukaryotic translation initiation factor 4E (PveIF4E) is the putative functional determinant of this resistance. The objective of this research was to expand the characterization of ClYVV resistance in common bean through greenhouse‐based phenotypic evaluation of 391 accessions of the USDA P. vulgaris Core Collection, a collection of 99 snap bean cultivars and breeding lines, and an extended host differential panel of 75 genotypes with known response to other viral pathogens and to determine if previously designed Kompetitive Allele Specific Polymerase Chain Reaction (KASP) assays could be validated for future bc‐3 resistance allele mining. As a result, one accession from the Core Collection, one additional snap bean breeding line, and 19 dry bean genotypes representing most major market classes of common bean were identified to be resistant. All genotypes that remained symptomless throughout the evaluation were demonstrated to possess mutations in PveIF4E that corresponded with either bc‐3 or bc‐32. In addition, the bc‐u bc‐22 allele combination and the By‐2 allele were also demonstrated to condition resistance phenotypes to ClYVV. These results synthesize a new model for genetic resistance to ClYVV and validate the use of the KASP assays for large‐scale bc‐3 allele mining.

Development and validation of a quantitative real-time polymerase chain reaction assay specific for the detection of Rickettsia felis and not Rickettsia felis-like organisms.

Human infections with Rickettsia felis have been reported worldwide. Recent studies have revealed the presence of many closely related but unique rickettsiae, referred to as Rickettsia felis-like organisms (RFLO), identified in various arthropods. Due to the recent discovery of the lack of specificity of earlier R. felis-specific assays, there has become a need to develop a new generation of R. felis-specific molecular assays that will differentiate R. felis not only from other rickettsiae but more importantly from other members of the R. felis genogroup that may not be pathogenic to humans. This new generation of assays is essential for determining the true risk for flea-borne spotted fever (FBSF) by surveying arthropod vectors/hosts. Because of the lack of specificity of previous assays developed to detect R. felis infections, prior surveys may have overestimated the prevalence of R. felis in arthropod vectors and thus the perceived risk of FBSF. We have developed a specific quantitative real-time polymerase chain reaction (qPCR) assay to detect R. felis (RfelB). Specificity of the assay was determined by testing it with a panel of 17 related Rickettsia species and 12 nonrickettsial bacterial DNA preparations. The RfelB qPCR assay was positive for R. felis DNA and negative for all of the 17 related Rickettsia species and 12 nonrickettsia bacterial DNA preparations. The limit of detection of the RfelB qPCR assay was determined to be two copies (two genoequivalents) per microliter of R. felis target ompB fragment-containing plasmid. Validation of the RfelB qPCR assay was accomplished by testing 83 previously sequence-confirmed R. felis and RFLOs containing DNA preparations from human and flea samples collected from different geographical locations around the world. This assay will be useful for rapid detection, identification, and enumeration of R. felis, an emerging human pathogen of worldwide importance, from both clinical and environmental samples.

Fine spatial structure of genetically distinct picocyanobacterial populations across environmental gradients in the Costa Rica Dome

We investigated the spatial variability of picocyanobacterial community structure across the Costa Rica Dome (CRD), an offshore upwelling system characterized by high seasonal abundance of Synechococcus spp. We constructed clone libraries of the rpoC1 gene to survey picocyanobacterial diversity and developed specific real‐time quantitative polymerase chain reaction assays to assess the distribution of genetically distinct Synechococcus (SYN) and Prochlorococcus (PRO) populations across vertical and horizontal physicochemical gradients. Flow cytometry data showed that cell abundances for both SYN and PRO were highest near the dome center. Phylogenetic analysis of rpoC1 sequences revealed a remarkably high and distinctive picocyanobacterial diversity (FLU1‐3, CRD1, Clade II, XV, XVI) that included “novel” SYN and PRO genotypes. Furthermore, genetically different populations exhibited vertical and horizontal spatial partitioning. Abundances of distinct SYN genotypes peaked at subsequent depth horizons, leading to a fine vertical structure with at least three populations stacked within the upper 30–40 m at the dome. Clade II and FLU1A peaked in surface waters, while maximum concentrations of CRD1, FLU1B, and Clade XVI occurred in the upper and lower thermocline, respectively. Horizontally, Clade II abundance in surface waters remained high across the entire region, while SYN genotypes CRD1 and FLU1A increased with shoaling of the thermo‐ and nutricline toward the center of the dome to become the dominant genotypes of the SYN assemblage in the dome. Below the mixed layer, Clade XVI and PRO genotype FLU2, virtually absent outside the dome, became abundant components of the picocyanobacterial assemblage. Despite their phylogenetic relatedness, FLU1A and FLU1B subclades followed different distributional patterns, suggesting ecological significance of the microdiversity within the clade. The unprecedented fine vertical structure demonstrated for SYN genotypes is driven by sharp physicochemical gradients (e.g., density, nutrient, oxygen, and trace metals) created by the dome and the presence of a shallow oxycline that enhances habitat diversification.

Detection of intrahepatic total and covalently closed circular DNA by a sensitive and specific quantitative PCR assay and its clinical significance

Background: CccDNA serves as a template for the production of HBV pregenomic-RNA and shows viral replication activity. It is responsible from the persistence of HBV during antiviral therapy. We report total-DNA and cccDNA levels in the liver biopsy samples of 6 chronic hepatitis B(CHB) patients during different phases of undergoing antiviral therapy by a sensitive and specific quantitative real-time polymerase chain reaction(PCR) assay. Methods & Materials: All the patients had undetectable serum HBV-DNA at the time of biopsy. Each patient underwent a liver biopsy, and a small piece of the biopsy specimens at least 2 mm in length was sent to labaratory in 0.9% NaCl solution. DNA was extracted from tissue using Alkaline phenol-chloroform- izoamyl alcohol process. PCR amplification was performed by TaqMan real-time PCR method. Primers and probes for intrahepatic total-DNA, cccDNA and lamivudine-resistance were designed using Oligoware 1.0 software program. ABI PRISM 7700 Sequence Detection System was used for all the analysis. Results: First patient was recieving lamivudine(LAM) for 5 years. Second and third patient recieving LAM for 38 and 42 months had elevated transaminase for the last 14 and 18 months despite undetectable serum HBV-DNA. Fourth patient was recieving LAM add-on adefovir dipivoxil for 63 months following 5 years of LAM. Fifth patient was recieving LAM add-on tenofovire disoproxil fumarat(TDV) for 3 months following 9 years of LAM. Sixth patient was recieving TDV for 3 months following one year of pegylated-IFN. Intrahepatic total\cccDNA levels in patients were 5,6x106\6,4x104, 8,8x107\4x104, 4,9x106\9,5x104, 5,5x106\2,7x104, 5,1x103\-, 9,2x107\1,1x104 coppies\mg.tissue, respectively. Although all the patients except first and second had normal levels of serum transaminase as well as undetectable serum HBV-DNA at the time of biopsy, YMDD mutations were identified in all except fourth and sixth one, then treatment regimens were reconstructed. Conclusion: Our results show that cccDNA persists in hepatocyte nucleus, even in with serological evidence of viral clearance under the long-term antiviral theraphy and its detection provides valuable information for the emergence of drug-resistance to manage treatment early. Investigating the intrahepatic forms of HBV-DNA is a good implementation for evidence-based medicine as an alternative in clinical practice. Therefore it is important to develop standardized assays to be used on clinical samples.

Cutaneous and visceral leishmaniasis co-infection in dogs from Rio de Janeiro, Brazil: evaluation by specific PCR and RFLP-PCR assays.

During a diagnostic evaluation of canine visceral leishmaniasis (VL), two of seventeen dogs were found to be co-infected by Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi. Specific polymerase chain reaction (PCR) and restriction fragment length polymorphism-PCR (RFLP-PCR) assays were performed. PCR assays for Leishmania subgenus identification followed by RFLP-PCR analysis in biopsies from cutaneous lesions and the spleen confirmed the presence of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi in those fragments. This report reinforces the importance of using serological and molecular techniques in the epidemiological surveillance of canine populations in endemic areas in which both diseases are known to co-exist. In such cases, a reassessment of the control measures is required.

Isolation, molecular identification and antibiogram ofPasteurella multocidain haemorrhagic septicaemia outbreak of buffaloes in West Bengal

Pasteurella multocida was isolated from clinically ill buffaloes in an outbreak of haemorrhagic septicaemia (HS). The isolates were identified by conventional culture along with molecular method of identification by polymerase chain reaction (PCR). An expected PCR amplicon of ∼460 bp and ∼620 bp products was observed using species specific PM-PCR, capsular type B specific PCR assays, respectively. In Kirby-Bauer disc diffusion method, the isolates were found to be sensitive to ofloxacin, ceftriaxone, cotrimoxazole, amikacin, gentamicin, intermediately sensitive to ciprofloxacin and resistant to tetracycline, ampicllin-salbactam, azithromycin, ceftazidime. PCR assays were found simple and quick diagnostic approach of haemorrhagic septicaemia and P. multocida capsular type B was found resistant to many antimicrobials.

Burkholderia pseudomallei in soil samples from an oceanarium in Hong Kong detected using a sensitive PCR assay

Melioidosis, caused by Burkholderia pseudomallei, is an emerging infectious disease with an expanding geographical distribution. Although assessment of the environmental load of B. pseudomallei is important for risk assessment in humans or animals in endemic areas, traditional methods of bacterial culture for isolation have low sensitivities and are labor-intensive. Using a specific polymerase chain reaction (PCR) assay targeting a Tat domain protein in comparison with a bacterial culture method, we examined the prevalence of B. pseudomallei in soil samples from an oceanarium in Hong Kong where captive marine mammals and birds have contracted melioidosis. Among 1420 soil samples collected from various sites in the oceanarium over a 15-month period, B. pseudomallei was detected in nine (0.6%) soil samples using bacterial culture, whereas it was detected in 96 (6.8%) soil samples using the specific PCR assay confirmed by sequencing. The PCR-positive samples were detected during various months, with higher detection rates observed during summer months. Positive PCR detection was significantly correlated with ambient temperature (P<0.0001) and relative humidity (P=0.011) but not with daily rainfall (P=0.241) or a recent typhoon (P=0.787). PCR-positive samples were obtained from all sampling locations, with the highest detection rate in the valley. Our results suggest that B. pseudomallei is prevalent and endemic in the oceanarium. The present PCR assay is more sensitive than the bacterial culture method, and it may be used to help better assess the transmission of melioidosis and to design infection control measures for captive animals in this unique and understudied environment.

Detection of Toxoplasma gondii in shellfish and fish in parts of China

In this study, a total of 3432 aquatic animals covering eight species were tested by specific polymerase chain reaction (PCR) assays to investigate the presence of Toxoplasma gondii, in China. The results showed that, out of 618 Procambarus clarkii samples collected from the different areas in southeast China, four samples from Jiangxi province were positive. Of 456 Hypophthalmichthys molitrix samples, one sample from Jiangsu province was positive. In addition, there was only one positive sample collected from Shandong province out of 813 Macrobranchium nipponense samples. All other samples, including 309 Cyprinus carpio, 398 Concha Ostreae, 98 Momopterus albus, 426 Penaeus monodon Fabricius and 309 Carassius auratus were negative. The results suggested that the level of T. gondii in aquatic animals was low in China. However, the importance of T. gondii in aquatic animals should not be ignored. Consumption of contaminated raw shellfish may represent a considerable health threat.

DETECTION OF HUMAN INTESTINAL PROTOZOA BY USING MULTIPLEX ALLELE SPECIFIC POLYMERASE CHAIN REACTION (MAS-PCR) In New Damietta City

Enteric protozoa continue to be the most commonly encountered parasitic diseases causing significant morbidity and mortality in developing regions of the world affecting millions of people. This study assessed the use of Multiplex Allele Specific Polymerase Chain Reaction (MAS-PCR) assay and microscopy for detection and identification of common pathogenic protozoan parasites in New Damietta city of North Delta region, Egypt. During Jun 2013 until Sept 2013, fresh stool samples were obtained from 249 patients up to 65 years of age attending the internal clinic of the Damietta University Hospital and those visiting their general practitioner (GP) of outpatient clinics because of gastrointestinal symptoms. Stool samples collected was preserved at -200C for DNA extraction whilst the remaining was preserved in sodium acetate-acetic acid formalin and concentrated using the formol-ether technique for microscopic examination. DNA extracts were analyzed with the multiplex allele specific Polymerase Chain Reaction (MAS-PCR) for pathogenic protozoan parasites. The diagnostic results obtained using a multiplex allele specific PCR for the detection of E. histolytica/dispar, G. lamblia and C. parvum/C. hominis were compared with these obtained by routine microscopy of faecal samples from patients. 69 samples were positive by MAS-PCR assays, 9 cases of G. intestinalis infection, 34 cases of D. fragilis infection, 3 cases of E. histolytica infection, 17 cases E.dispar and 6 cases of Cryptosporidium infection in the clinical samples. By microscopy, only 32 samples were positive for one or more of the enteric protozoa, 5cases of G. intestinalis infection, 9cases of D. fragilis infection, 13 cases of E. histolytica infection, and 1 cases of Cryptosporidium infection in the clinical samples. However, there are no cases of E.dispar observed. Mixed infections were detected in 4 samples. The sensitivities varied from 58% for D. fragilis to 47% for E. histolytica, 35% for Giardia, and 30% for Cryptosporidium, while the specificities also varied from 97% for E. histolytica to 99% for D. fragilis and 100% for E.dispar . No cross-reactivity was detected in stools samples containing various bacterial, viral, and other protozoan species. This present study showed relatively high rates of protozoa infections in the study patients. The study has also demonstrated that the multiplex real time PCR assay was more sensitive compared to microscopy in the diagnosis of the intestinal protozoa parasites and thus, molecular methods must be considered the diagnostic methods of choice for enteric protozoan parasites. Keywords: Human intestinal protozoa, Stools specimens, Microscopy, Multiplex Allele Specific Polymerase Chain Reaction (MAS-PCR), Diagnosis.

DETECTION AND DIFFERENTIATION OF HUMAN PROTOZOAN PARASITES IN STOOL SPECIMENS BY USING MULTIPLEX ALLELE SPECIFIC POLYMERASE CHAIN REACTION (MAS-PCR) In New Damietta City, Egypt

Enteric protozoa continue to be the most commonly encountered parasitic diseases causing significant morbidity and mortality in developing regions of the world affecting millions of people. This study assessed the use of Multiplex Allele Specific Polymerase Chain Reaction (MAS-PCR) assay and microscopy for detection and identification of common pathogenic protozoan parasites in New Damietta city of North Delta region, Egypt. Between Jun 2013 until Sept 2013, fresh stool samples were obtained from 249 patients up to 65 years of age attending the internal clinic of the Damietta University Hospital and those visiting their general practitioner (GP) of outpatient clinics because of gastrointestinal symptoms. Stool samples collected was preserved at -200C for DNA extraction whilst the remaining was preserved in sodium acetate-acetic acid formalin and concentrated using the formol-ether technique for microscopic examination. DNA extracts were analyzed with the multiplex allele specific Polymerase Chain Reaction (MAS-PCR) for pathogenic protozoan parasites. The diagnostic results obtained using a multiplex allele specific PCR for the detection of E. histolytica/dispar, G. lamblia and C. parvum/C. hominis were compared with these obtained by routine microscopy of faecal samples from patients. 69 samples were positive by MAS-PCR assays , 9 cases of G. intestinalis infection, 34 cases of D. fragilis infection, 3 cases of E. histolytica infection, 17 cases E.dispar and 6 cases of Cryptosporidium infection in the clinical samples. By microscopy, only 32 samples were positive for one or more of the enteric protozoa, 5cases of G. intestinalis infection, 9cases of D. fragilis infection, 13 cases of E. histolytica infection, and 1 cases of Cryptosporidium infection in the clinical samples. However, there are no cases of E.dispar observed. Mixed infections were detected in 4 samples. The sensitivities varied from 58% for D. fragilis to 47% for E. histolytica, 35% for Giardia, and 30% for Cryptosporidium, while the specificities also varied from 97% for E. histolytica to 99% for D. fragilis and 100% for E.dispar . No cross-reactivity was detected in stool samples containing various other bacterial, viral, and protozoan species. This present study showed relatively high rates of protozoa infections in the study patients. The study has also demonstrated that the multiplex real time PCR assay was more sensitive compared to microscopy in the diagnosis of the intestinal protozoa parasites and thus, molecular methods must be considered the diagnostic methods of choice for enteric protozoan parasites. Keywords: Human intestinal protozoa, Stool specimens, Microscopy, Multiplex Allele Specific Polymerase Chain Reaction (MAS-PCR), Diagnosis.

Development and evaluation of specific PCR and LAMP assays for the rapid detection of Phytophthora melonis

Phytophthora melonis is a widespread and devastating pathogen for the Cucurbitaceae family. Early and accurate detection of P. melonis is essential to control the disease in the field. To establish a simple, visual, and rapid detection system for P. melonis, we developed nested polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) systems based on the Ras-related protein (Ypt1) gene. All 36 isolates of P. melonis, from geographically distinct counties in China, yielded positive detection results on LAMP or nested PCR assays. No cross reaction was observed with other oomycetes or fungal pathogens. A sensitivity assay showed that both methods had a detection limit of 10 fg genomic DNA. We also detected P. melonis in diseased cucumber tissues and soils, and evaluated positive detection rates using LAMP, nested PCR, and conventional isolation methods. The results suggest that the LAMP assay has the greatest potential for active detection of P. melonis in regions that are at risk of contracting the disease, and for use in resource-poor settings.

Difficulties in diagnosing terminal ileitis due to Yersinia pseudotuberculosis

We report three patients with terminal ileitis and positive fecal cultures with Yersinia pseudotuberculosis. From one patient, a virulence plasmid (pYV)-negative Y. pseudotuberculosis was isolated, which represents the second finding of a pYV-negative isolate associated with human disease. All patients were treated with ciprofloxacin and fully recovered. Since conventional culture methods for yersiniosis are gradually replaced with molecular tests not recognizing Y. pseudotuberculosis, we recommend to include a specific culture medium or to apply a specific polymerase chain reaction (PCR) assay on fecal samples from patients suspected of terminal ileitis.

Isolation of Bluetongue virus from canine abortions

Three aborted canine fetuses were submitted to the Animal Health Diagnostic Center at Cornell University in November 2011 and September 2012 for diagnostic workups to determine the causes of the reproductive difficulties. Histological assessments of the sampled tissues were inconclusive due to the autolysis. Tests to detect bacterial causes of the abortions were also negative. Virus isolation testing on pooled tissues from the fetuses identified a cytopathogenic agent in cell cultures. Fluorescent antibody tests on the infected cells gave a positive reaction for Bluetongue virus, and subsequent serotype specific reverse transcription polymerase chain reaction assays identified the isolates as Bluetongue virus serotype 11. The current report describes the isolation of Bluetongue virus from dogs unrelated to contaminated vaccines and in the absence of a raw meat diet.

Role of the Central Venous Catheter in Bloodstream Infections Caused by Coagulase-negative Staphylococci in Very Preterm Neonates

Pathogenesis of coagulase-negative staphylococcal bloodstream infections among preterm neonates is debated: central venous catheters (CVCs) are considered the major cause and the cornerstone of prevention measures. The role of other means of transmission is unknown. We developed a specific quantitative polymerase chain reaction assay targeting the dnaJ gene from Staphylococcus epidermidis and Staphylococcus capitis to detect DNA from CVC used in preterms. Performance of the polymerase chain reaction was tested against 2 control groups of CVC yielding positive (n = 24) or negative (n = 63) conventional cultures. We also explored retrospectively the DNA load of CVC having a negative conventional bacterial culture and obtained from 34 very preterm neonates with catheter-related bloodstream infections (CR-BSIs) established by usual clinical and biologic criteria. The molecular approach allowed detection of corresponding DNA from all the positive control catheters. Among the 34 episodes of CR-BSI yielding a negative conventional CVC culture, 8 (23.5%) had a positive polymerase chain reaction signal (5 S. epidermidis and 3 S. capitis). This percentage did not significantly differ according to the staphylococcal species, the delay between the CVC insertion and the beginning of the sepsis or between the blood culture collection and the CVC removal. These results conform to the previously published 70% of CR-BSI for whom the origin could be questioned. CVC removal in preterms is often performed in cases of CR-BSI; our study supports the hypothesis that in some cases the responsibility of CVC is questionable.