Multiplex Polymerase Chain Reaction For Detection Research Articles

Development of a multiplex real-time quantitative reverse-transcription polymerase chain reaction for the detection of four bee viruses

Viruses in the families Dicistroviridae and Iflaviridae are among the main threats to western honey bees (Apis mellifera) and native bee species. Polymerase chain reaction (PCR) is the gold standard for pathogen detection in bees. However, high throughput screening for bee virus infections in singleplex PCR reactions is cumbersome and limited by the high quantities of sample RNA required. Thus, the development of a sensitive and specific multiplex PCR detection method for screening for multiple viruses simultaneously is necessary. Here, we report the development of a one-step multiplex reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay to detect four viruses commonly encountered in pollinator species. The optimized multiplex RT-qPCR protocol described in this study allows simultaneous detection of two dicistroviruses (Israeli acute paralysis virus and Black queen cell virus) and two iflaviruses (Sacbrood virus and Deformed wing virus) with high efficiency and specificity comparable to singleplex detection assays. This assay provides a broad range of detection and quantification, and the results of virus quantification in this study are similar to those performed in other studies using singleplex detection assays. This method will be particularly useful for data generation from small-bodied insect species that yield low amounts of RNA.

Establishment of a multiplex polymerase chain reaction detection assay for three common harmful microalgae in the East China Sea.

It is urgent to develop techniques that can simultaneously detect multiple microalgae, due to the diversity of harmful algal blooms (HABs)-forming algal species. The target algae species in this study are Heterosigma akashiwo, Prorocentrum donghaiense and Karenia mikimotoi. These algae are the dominant species that cause HABs in the East China Sea, and the multiple detection technique focusing on these three algae is not common. Therefore, this study established a multiplex polymerase chain reaction(mPCR) to diagnose the three algae, which is simple and low cost. First, the corresponding specific primers were designed based on the D1-D2 region of the large subunit (LSU) ribosomal DNA sequence. Then, mPCR was established and the reaction conditions were optimized. And the specificity, sensitivity, and stability of mPCR were evaluated. The result of specificity test showed that the established mPCR had good specificity for the target microalgae and did not cross-react with eighteen non-target microalgae. The sensitivity of experiment was 3.3 × 10-1ng μL-1, and the established mPCR was not affected by the interfering microalgae. Moreover, the practicability evaluation of mPCR by using the simulated natural water samples showed that the detection limit of target microalgae was 100 cells mL-1, which could meet the demand for early warning of HABs. In summary, the established mPCR is characterized by strong specificity, good stability, and multiple analysis to detect H. akashiwo, P. donghaiense, and K. mikimotoi.

Establishment of a multiplex-PCR detection method and development of a detection kit for five animal-derived components in edible meat

A multiplex polymerase chain reaction (PCR) detection method for the simultaneous detection of animal-derived components from deer, cow, sheep, pig and horse in edible meat was established, and a multiplex PCR detection kit for the rapid detection of animal-derived components was developed. According to the mitochondrial cytochrome b (Cyt b) gene of bovine species, sheep species, pig species and horse species and the mitochondrial cytochrome c oxidase subunit I (COX 1) gene of sika deer and red deer as the target gene sequences of primers, the specific primers of five different species were designed, the PCR system was optimized, and the multiplex PCR identification method of five animal-derived components was established. The minimum detection amount was determined by sensitivity test. The results showed that five meat specific amplification bands could be found at the same time in the same reaction system, including 173 bp fragment for venison, 148 bp for beef, 261 bp for pork, 100 bp for mutton and 424 bp for horse, indicating that the method is specific and stable. The minimum detection limit by this method was 1 ng/μL, showing a high sensitivity. According to the different sites in different areas of animal mitochondrial genes, a multiplex PCR detection method was established and a detection kit was developed, and the rapid, sensitive, stable and high-throughput detection of five animal-derived components and adulterated animal components in edible meat can be realized by using the kit.

A diagnostic test of real-time PCR detection in the diagnosis of clinical bloodstream infection

Blood culture remains the standard for diagnosing bloodstream infections, but it is difficult to identify bacteria directly and timeliness. The real-time polymerase chain reaction (PCR) has the potential to fill this diagnostic gap. This study intends to explore the sensitivity and specificity of PCR in detecting bloodstream infection pathogens and to compare it with routine blood culture to explore its clinical application value. A total of 126 patients with bloodstream infections collected from various clinical departments of The First Hospital of Hebei Medical University. The patient's sample was divided into two parts. The one for multiplex PCR detection was performed using the Pathogeno Elite Multiplex PCR kit. Another blood culture was a fully automatic blood culture system from Autobio company. Among the 126 patients, a total of 17 pathogens were detected by PCR and blood culture both methods. PCR detected a total of 43 positive samples and 83 negative samples. Five samples were positive with blood culture, and 81 were negative. The negative predictive value of PCR was 0.98, with a sensitivity of 0.71 and a specificity of 0.68. A total of 38 specimens were positive for PCR but negative for blood culture, and 2 samples were positive for blood culture but negative for PCR. The top 5 pathogens with PCR detection were Epstein-Barr virus (27 cases), Human herpes virus 5 (9 cases), Klebsiella pneumoniae (5 cases), Staphylococcus (5 cases), and Stenotrophomonas maltophilia (4 cases). PCR detection can rapidly identify more pathogens and even multi-pathogen infections. Therefore, PCR testing may improve pathogen detection in patients with suspected bloodstream infections, enabling targeted treatment of patients.

Multiplex polymerase chain reaction detection of Streptococcus pneumoniae and Haemophilus influenzae and their antibiotic resistance in patients with community-acquired pneumonia from southwest Iran

BackgroundThis study aimed to evaluate the occurrence of Streptococcus pneumoniae and Haemophilus influenzae in sputum of patients with community-acquired pneumonia (CAP) using culture and multiplex polymerase chain reaction (M-PCR) methods and to survey the antibiotic resistance patterns of aforesaid isolates.ResultIn total, 23.9 % (n = 22/92) of sputum samples showed positive results in the culture method. S. pneumoniae and H. influenzae were isolated from 15 (16.3 %) and 7 (7.6%) samples, respectively. Using M-PCR, 44 (47.8 %) samples were positive for S. pneumoniae and H. influenzae. Of these, S. pneumoniae and H. influenzae were detected in 33 (35.8%) and 11 (11.9%) of the sputum samples, respectively. The sensitivity, specificity, and accuracy rates of PCR in detection of S. pneumoniae in comparison with culture method were 100, 76.6, and 83.6%, respectively. While, the sensitivity, specificity, and accuracy rates of PCR in detection of H. influenzae in comparison with culture method were 100, 95.3, and 95.8%, respectively. Out of 11 isolates of H. influenzae, two strains confirmed as H. influenzae type b (Hib) and 3 isolates were type f. However, 6 isolates were non-typable. The co-trimoxazole and amoxicillin/clavulanate were the less effective antibiotics against S. pneumonia and H. influenzae, respectively. Ceftriaxone with 13.3% resistance rates was the most effective antibiotic against S. pneumoniae, while, clarithromycin, ceftriaxone, and gentamicin with resistance rates of 28.6% for each one were the most effective chemicals against H. influenzae isolates.ConclusionIn this study, the prevalence of S. pneumoniae was more than H. influenzae using culture and M-PCR methods. The M-PCR provided better efficiency in detecting the bacterial agents in CAP patients compared to culture method. This method can improve the early detection of pathogens contributed to CAP. The drug resistant S. pneumoniae and H. influenzae indicated the need to develop a codified monitoring program to prevent further spread of these strains.

Establishment and Preliminary Application of Multiplex Fluorescent Quantitative PCR for Simultaneous Detection of BVDV, BRV and BCV

Background: In this study, we aimed to establish a multiplex fluorescence quantitative polymerase chain reaction (PCR) method for the identification and detection of bovine viral diarrhea virus (BVDV), bovine rotavirus (BRV) and bovine coronavirus (BCV). Methods: Based on the highly conserved sequences of BVDV E2 gene, BRV VP6 gene and BCV N gene in GenBank, specific primers were designed to amplify the target gene fragments of each virus and the reaction conditions and system were optimized. Multiple fluorescence quantitative methods were established by fluorescence quantitative PCR. Result: The minimum detection limits of plasmid standards for BVDV, BRV and BCV by multiplex fluorescence quantitative PCR were 1.19×102 copies/μL, 3.89×101 copies/μL and 3.74×101 copies/μL, respectively. The lowest sensitivity of the established method was 100 times higher than that of conventional PCR and had high sensitivity. Furthermore, BVDV, BRV and BCV were amplified specifically, with no cross-reactivity with Escherichia coli (E. coli), Salmonella and infectious bovine rhinotracheitis virus (IBRV). The intra-and inter-group coefficients of variation were less than 1%, showing good assay repeatability. Using the established method and ordinary multiplex PCR to simultaneously detect 150 clinical diarrheal disease material samples, the coincidence rate of samples with mixed infection of the three viruses was 83.3%. The results showed that the multiplex fluorescent quantitative PCR detection method established in this study provides a rapid, sensitive and specific technique for clinical diagnosis and epidemiological monitoring of BVDV, BRV and BCV.

A rapid and accurate method for the detection of four aminoglycoside modifying enzyme drug resistance gene in clinical strains of Escherichia coli by a multiplex polymerase chain reaction.

BackgroundAntibiotics are highly effective drugs used in the treatment of infectious diseases. Aminoglycoside antibiotics are one of the most common antibiotics in the treatment of bacterial infections. However, the development of drug resistance against those medicines is becoming a serious concern.AimThis study aimed to develop an efficient, rapid, accurate, and sensitive detection method that is applicable for routine clinical use.MethodsEscherichia coli was used as a model organism to develop a rapid, accurate, and reliable multiplex polymerase chain reaction (M-PCR) for the detection of four aminoglycoside modifying enzyme (AME) resistance genes Aac(6′)-Ib, Aac(3)-II, Ant(3″)-Ia, and Aph(3′)-Ia. M-PCR was used to detect the distribution of AME resistance genes in 237 clinical strains of E. coli. The results were verified by simplex polymerase chain reaction (S-PCR).ResultsResults of M-PCR and S-PCR showed that the detection rates of Aac(6′)-Ib, Aac(3)-II, Ant(3″)-Ia, and Aph(3′)-Ia were 32.7%, 59.2%, 23.5%, and 16.8%, respectively, in 237 clinical strains of E. coli. Compared with the traditional methods for detection and identification, the rapid and accurate M-PCR detection method was established to detect AME drug resistance genes. This technique can be used for the clinical detection as well as the surveillance and monitoring of the spread of those specific antibiotic resistance genes.

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.

Multiplex polymerase chain reaction detection of Shiga toxin genes and antibiotic sensitivity of Escherichia coli O157:H7 isolated from beef meat in Quetta, Pakistan

AbstractThe objective of this study was to conduct a qualitative analysis of raw beef meat sold in the city of Quetta, Pakistan for presence and drug sensitivity of the potentially pathogenic Escherichia coli strain O157:H7. The study used 200 raw beef meat samples collected from retail butcher shops. Conventional and rapid biochemical tests, latex agglutination and multiplex polymerase chain reaction (PCR) using primers designed for the rfb O157 and flic H7 genes were used to detect E. coli O157:H7. All O157:H7 isolates were also tested for Shiga toxin genes stx 1 and stx 2. The prevalence of E. coli O157:H7 in collected beef meat samples was 10%. Detection through PCR was found more sensitive than detection of O and H antigens. The quantity of E. coli O157:H7 isolates positive for Shiga toxins was 50% (20% for stx 1, 45% for stx 2 and 10% for both stx 1 and stx 2). Season wise variation showed highest E. coli O157:H7 prevalence during summer months. A further concern is that E. coli O157:H7 isolates were resistant to a range of common antibiotics. The results indicate an urgent need for applying proper food hygiene practices in the Quetta region to reduce incidence of foodborne diseases and they also emphasize the global problem of antimicrobial resistance.Practical applications E. coli O157:H7 is as a potentially threatening foodborne pathogen. A significant prevalence of E. coli O157:H7 detected in raw beef meat from retail outlets in the city of Quetta indicates an urgent need for applying proper food hygiene practices in the Quetta region to reduce the incidence of foodborne diseases. Furthermore, resistance of the E. coli O157:H7 isolates to a range of commonly used antibiotics emphasizes the global problem of antimicrobial resistance. The multiplex PCR method used here is a reliable, sensitive, and relatively rapid technique for detecting E. coli O157:H7 in food and environmental samples and important for ongoing surveillance to minimize contamination of raw meat products and associated cross contamination by E. coli O157:H7.

Simultaneous detection of Theileria annulata and Theileria orientalis infections using recombinase polymerase amplification

Theileriosis is a disease of domesticated animals in tropical and subtropical countries and causes significant reductions in livestock productivity. The arid region of Punjab in Pakistan is notorious for the presence of the vector tick (Acari: Ixodidae) and tick-borne diseases, such as theileriosis and babesiosis. The distribution of Theileria annulata and T. orientalis in the Chakwal district of Punjab was determined by developing a multiplex recombinase polymerase amplification (RPA) assay as a scientific basis for formulating control strategies for bovine theileriosis. Specificity was evaluated using DNA from related piroplasm species, while analytical sensitivity was calculated using a long fragment of the enolase gene. A total of 188 blood samples were collected on FTA cards (Whatman®) from tick-infested asymptomatic breeds of cattle (Bos indicus, Bos taurus, and Bos indicus × Bos taurus) in the study district. Finally, infections with of T. annulata and T. orientalis were detected using the multiplex RPA and compared with the conventional multiplex polymerase chain reaction (PCR). The multiplex RPA specifically amplified 282-bp and 229-bp fragments of the enolase gene from T. annulata and T. orientalis and had no cross-reaction with other piroplasm species. It was determined that 45 (23.9%) and 5 (2.6%) out of 188 blood samples were positive for T. annulata and T. orientalis, respectively, when examined using RPA. Multiplex PCR detection indicated that 32 (17.0%) and 3 (1.6%) blood samples were positive for T. annulata and T. orientalis, respectively. In the present study, a specific RPA method was developed for simultaneous differentiation and detection of T. annulata and T. orientalis infections and used for the first time for the detection of the two bovine Theileria infections.

Multiplex polymerase chain reaction detection of selected bacterial species from symptomatic and asymptomatic non-vital teeth with primary endodontic infections.

The aim of the present study was to investigate the presence of selective anaerobic microorganisms in primary root canal infections of symptomatic and asymptomatic non-vital teeth with periapical pathosis using multiplex polymerase chain reaction. A total of 100 root canal samples (50 from symptomatic and 50 from asymptomatic teeth) were obtained from patients with primary endodontic infections. DNA extracted from the samples was amplified by using specific primers for the 16S rRNA gene of each bacterium, and semiquantification was done to analyze the prevalence of microorganisms and their correlation to clinical features. Treponema denticola (T.denticola) was present in 21 (42%) and 29 (58%) samples in the symptomatic and asymptomatic groups, respectively. Tannerella forsythia, Porphyromonas gingivalis (P.gingivalis), and Fusobacterium nucleatum (F.nucleatum) were significantly high (P<.05) in the symptomatic group, whereas Prevotella intermedia was significantly high (P<.05) in the asymptomatic group. The mean counts of T.denticola and F.nucleatum were significantly high (P<.05) in the symptomatic group. For symptoms, P.gingivalis, T.denticola, and F.nucleatum were significantly associated with clinical features. Significant differences exist in the bacterial composition between asymptomatic and symptomatic primary endodontic infections. As well as presence of pathogens, other factors, such as the phenotypic trait of bacteria and interactions among bacterial members, might play a determining role in the pathogenicity of primary endodontic infections.

Nested polymerase chain reaction targeting 16S rRNA gene in diagnosis of acute bacterial meningitis

CONTEXT: Diagnosing acute bacterial meningitis (ABM) among children presenting to tertiary health care settings is often difficult because of prior administration of antimicrobials. AIMS: The present study was an attempt to diagnose cases of ABM in children with the help of nested polymerase chain reaction (PCR). SETTINGS AND DESIGN: It is a prospective observational study, in which a total of 84 clinically suspected cases and cerbrospinal fluid (CSF) biochemical parameters suggestive of ABM were included in the study. METHODS AND MATERIAL: CSF samples were subjected to Gram staining, bacterial culture and biochemical identification tests as well as panbacterial nested PCR targeting 16S rRNA gene sequence. Subsequently, a nested multiplex PCR for detection of the three fastidious organisms, viz. Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae, was performed. STATISTICAL ANALYSIS USED: The sensitivity, specificity, PPV, NPV, LR+, and LR− of the tests were calculated. RESULTS: The sensitivity of Gram stain, bacterial culture, and nested PCR targeting 16SrRNA were observed to be 16.21%, 9.45%, and 97.29% respectively. Further, the NPV and LR− the PCR were found to be 83.33 and 0.02 respectively. Species specific nested multiplex PCR was able to detect S. pneumoniae (n = 7), N. meningitidis (n = 2) and H. influenzae (n = 1). CONCLUSIONS: The results indicates that nested PCR targeting 16S rRNA gene may be used in diagnosis of ABM. Further, nested multiplex PCR targeting the three important fastidious bacterial pathogens in ABM cases has showed their presence in our region.

Identifying virulence factor genes in E. coli in animal houses and their transmission to outside environments

Escherichia coli is a common bacterium in the air; it can affect the health of humans and animals. The carrying status of five primary virulence factor genes in E. coli (i.e., STa, STb, LTa, stx1, and stx2/stx2e) in various animal houses and their spread to the outside environment were investigated by comparing the air inside animal houses with that outside the houses. Multiplex polymerase chain reaction detection of the five genes was performed on samples from five chicken houses, and 117 E. coli isolates were obtained. From five swine houses, 120 were obtained, and from six cow houses, 143 were obtained. Results reveal that E. coli virulence genes derived from animal houses can be propagated via gas exchange from the inside to the outside and, depending on weather conditions, across some distance, resulting in biological pollution and the spread of pathogenic microorganisms. This study of the transmission of environmental microbial aerosols in animal houses has great significance in public health and epidemiology.

Rapid Detection and Identification of Mycotoxigenic Fungi and Mycotoxins in Stored Wheat Grain.

This study aimed to assess the occurrence of toxigenic fungi and mycotoxin contamination in stored wheat grains by using advanced molecular and analytical techniques. A multiplex polymerase chain reaction (PCR) strategy was established for rapid identification of mycotoxigenic fungi, and an improved analytical method was developed for simultaneous multi-mycotoxin determination in wheat grains by liquid chromatography-tandem mass spectrometry (LC/MS/MS) without the need for any clean-up. The optimized multiplex PCR method was highly specific in detecting fungal species containing species-specific and mycotoxin metabolic pathway genes. The method was applied for evaluation of 34 wheat grain samples collected from storage warehouses for the presence of mycotoxin-producing fungi, and a few samples were found positive for Fusarium and Aspergillus species. Further chemical analysis revealed that 17 samples contained mycotoxins above the level of detection, but only six samples were found to be contaminated over the EU regulatory limits with at least one mycotoxin. Aflatoxin B1, fumonisins, and deoxynivalenol were the most common toxins found in these samples. The results showed a strong correlation between the presence of mycotoxin biosynthesis genes as analyzed by multiplex PCR and mycotoxin detection by LC/MS/MS. The present findings indicate that a combined approach might provide rapid, accurate, and sensitive detection of mycotoxigenic species and mycotoxins in wheat grains.

Improved multiplex PCR primers for rapid identification of coagulase-negative staphylococci

Coagulase-negative staphylococci (CNS) are opportunistic pathogens that are currently emerging as causative agents of human disease. Though CNS are widespread in the clinic and food, their precise identification at species level is important. Here, using 16S rRNA sequencing, 55 staphylococcal isolates were identified as S. capitis, S. caprae, S. epidermidis, S. haemolyticus, S. pasteuri, S. saprophyticus, S. warneri, and S. xylosus. Although 16S rRNA sequencing is universally accepted as a standard for bacterial identification, the method did not effectively discriminate closely related species, and additional DNA sequencing was required. The divergence of the sodA gene sequence is higher than that of 16S rRNA. To devise a rapid and accurate identification method, sodA-specific primers were designed to demonstrate that species-specific multiplex polymerase chain reaction (PCR) can be used for the identification of CNS species. The accuracy of this method was higher than that of phenotypic identification; the method is simple and less time-consuming than 16S rRNA sequencing. Of the 55 CNS isolates, 92.72% were resistant to at least one antibiotic, and 60% were resistant to three or more antibiotics. CNS isolates produced diverse virulence-associated enzymes, including hemolysin (produced by 69.09% of the isolates), protease (65.45%), lipase (54.54%), lecithinase (36.36%), and DNase (29.09%); all isolates could form a biofilm. Because of the increasing pathogenic significance of CNS, the efficient multiplex PCR detection method developed in this study may contribute to studies for human health.

Rate of Detection of Multiple Organisms and Clostridium difficile with Stool Multiplex PCR Detection Test in Pediatrics

BackgroundNew multiplex molecular assays have been developed to determine the etiology of infectious gastroenteritis. Unfortunately, these assays can detect multiple organisms simultaneously along with Clostridium difficile (C.diff), making it difficult to differentiate true pathogen vs. colonization. In January 2015, our institution switched from traditional testing methods to a multiplex polymerase chain reaction (PCR) detection test (FilmArrayTM Gastrointestinal Panel. BioFireDX, Salt Lake City, Utah). The objective of our study was to determine the number of FilmArrayTMpanels that detected C.diff and/or multiple organisms.MethodsWe conducted a retrospective data review of FilmArray™ panels in pediatric patients 18 years and younger from January 2015 to December 2016. Stool samples were received from both inpatient and outpatient setting.ResultsIn 2016, 495 FilmArray™ panels were reviewed and 300 (61%) isolated at least one organism. Among the positives panels, 206 (69%) detected one organism, 73 (24%) detected 2 organisms and 21 (7.0%) detected 3 or more organisms. No more than 4 organisms were detected in a single panel. C.diff was most commonly isolated, found 105 times (25%), and 34 (31%) of these were in children younger than 2 years. Amongst the 105 C.diff isolates, 64% were alone and 35% with another organism. Amongst children younger than 2, C.diff was isolated alone in 13 (38%) samples and with another organism in 21 (62%) samples. In 2015, 353 panels were reviewed with a detection rate of 60.3%. C.Diff was isolated 70 times (24% of total isolates) and 22 (31%) were in children younger than 2 years. Amongst those C.diff isolates, 49% were alone and 51% with another organism. Amongst children younger than 2, C.diff was isolated alone in 8 (38%) samples and with another organism in 14 (62%) samples.ConclusionAlthough the FilmArray™ Gastrointestinal Panel is a useful single modality for determining the etiology of infectious gastroenteritis, more than one organism is frequently detected. C.diff has become the most common organism isolated among children at our institution. Caution should be used when interpreting the isolation of C.diff in younger children and when isolated with other organisms.Disclosures All authors: No reported disclosures.

Multiplex PCR for detection of water-borne bacteria

Microbial water-borne diseases still affect developing countries and are major water quality concerns throughout the world. Routine culture-based methods of identifying bacterial pathogens in water sources are laborious and time-consuming. Recently, the use of molecular techniques such as the polymerase chain reaction (PCR) has provided rapid and highly promising detection methods. In this study, we developed two multiplex PCR assays for simultaneous detection of six water-borne bacteria. Two triplex PCR protocols were developed to detect six target genes. The first protocol targets uidA (Escherichia coli), int (Shigella spp.), and gyrB (Pseudomonas aeruginosa) genes, while invA (Salmonella spp.), ompW (Vibrio cholera), and lacZ (coliforms) were amplified by the second protocol. Specificity testing was carried out for 12 reference strains. Furthermore, the applicability of the multiplex PCR assays for detection of these bacteria was investigated for 52 surface water samples. The results indicated that all primer pairs showed specificities only for their corresponding target organisms. The detection sensitivity of both multiplex PCR assays was 3 × 102 − 3 × 103 colony forming units. The developed assays represent simple and efficient diagnostic procedures for co-detection of water-borne bacteria and have the potential to provide earlier warnings of possible public health threats and more accurate surveillance of these organisms.

Molecular detection of Taenia spp. in dogs' feces in Zanjan Province, Northwest of Iran.

Aim:Echinococcus and Taenia spp. are important but neglected zoonotic helminths of dogs. Dogs as the most relevant definitive hosts harbor several species of Taenia and Echinococcus simultaneously in their gastrointestinal lumen which are morphologically indistinguishable. In this study, we used a multiplex polymerase chain reaction (PCR) method to identify Taeniid infections which seem to be highly distributed in the study region.Materials and Methods:A total of 450 dog fecal samples were collected from eight different areas of Zanjan province, northwest of Iran, and examined using a flotation method followed by multiplex PCR for detection and identification of parasites’ eggs.Results:Gastrointestinal parasites were found in 86 out of 450 fecal samples (19.1%) by microscopic examination. Taeniid eggs were observed in 5.6% of samples, containing 0.45%, 3.8%, and 1.3% Echinococcus granulosus, Taenia spp., and mix infection of both E. granulosus and Taenia spp., respectively. Echinococcus multilocularis was absent in the samples.Conclusion:A relatively low rate of E. granulosus (1.8%) was observed in this study. However, risks of this parasite should not be overlooked, and control programs need to be extended for this species and other Taeniid spp. In particular, dogs are recommended to be dewormed more frequently.

The epidemiological investigation of extended-spectrum β-Lactamases-producing Escherichia coli in clinical and environmental samples at the tumor hospital

Objective To analyze the genotypes of extended-spectrum β-Lactamases (ESBL)-producing Escherichia coli (E. coli) and its homology from the clinical and environmental samples at the tumor hospital. Methods The clinical samples (including the patient's blood, urine, sputum, hydrothorax, drainage fluid, stool, etc) and environmental samples (including the air, escort hand, door handles, bed rails, bedside table, medical staff hand) were collected at the Tumor Hospital Affiliated to Zhengzhou University from June 2012 to January 2013. The confirmatory test of ESBL phenotype was performed. β-Lactamase genes (blaTEM, blaSHV, blaOXA, blaCTX and blaFOX) were detected by multiplex polymerase chain reaction (PCR) assays. Pulsed-field gel electrophoresis (PFGE) was used to characterize the homology of all the strains. Results The phenotypic confirmatory test detected a total of 41 ESBL-producing E. coli, among which 36 stains were from clinical specimens (all were from 28 patients) and 5 strains from environmental specimens. Meanwhile, multiplex PCR detection of the genes of ESBL-producing E. coli was performed. TEM-1 or CTX-14 type was predominant in ESBL-producing E. coli carrying one gene. Those carrying two genes were mainly CTX-14 and TEM-1 genes, whereas three genes were CTX-14, TEM-1 and SHV-1. PFGE results showed that the specimens from 28 patients that located in gut or other clinical specimens shared no homology in ESBL-producing E. coli. However, the specimens from 3 patients and 1 patient's bedside table in the first surgical ward shared homology. Conclusions The major epidemic genes of E. coli in our hospital were TEM-1, CTX-14 and CTX-1. The same strain may have many genotypes, mainly with two genes. Exogenous infection may be one major way in ESBL-producing E. coli infection. Key words: Enterotoxigenic Escherichia coli; beta-Lactamases; Pulsed-field gel electrophoresis

Characterization of mating-type loci in rice false smut fungus Villosiclava virens

Ascospores of Villosiclava virens are a primary infection source of rice false smut. This phytopathogenic fungus exists in heterothallic form, and mating compatibility is regulated by mating-type locus 1 (MAT1). However, the MAT1 locus structure remains unknown. The MAT1-1 and MAT1-2 idiomorphs of V. virens were characterized and annotated on the basis of cDNA sequencing. A multiplex polymerase chain reaction (PCR) method was developed to identify the mating types of hyphae and sclerotia. The MAT1-1 locus of V. virens contains three mating-type genes: MAT1-1-1, MAT1-1-2 and MAT1-1-3, and a pseudogene similar to MAT1-2-1. The MAT1-2 locus harbors the MAT1-2-1 gene and a new mating-type gene MAT1-2-8. The mRNA of MAT1-1-1, MAT1-1-3 and MAT1-2-1, but not MAT1-1-2, was detectible by reverse transcription PCR in vegetative mycelia. However, the mRNA of MAT1-1-2 was detectible in the stroma, which is a sexual reproduction structure of V. virens. A multiplex PCR detection method was developed for the identification of the MAT1-1 and MAT1-2 idiomorphs. All 20 wild-type strains harbored either the MAT1-1 or MAT1-2 idiomorphs. Sclerotia that harbored both the MAT1-1 and MAT1-2 idiomorphs had potential to form fertile stromata, whereas those that harbored only the MAT1-1 idiomorph could not form mature stromata.