Modified multiplex PCR methods for comprehensive detection of Pectinatus and beer-spoilage cocci

Specific PCR primers were designed based on the 16S rRNA genes of recently proposed beer-spoilage species, Pectinatus haikarae, Megasphaera sueciensis, and M. paucivorans, and two sets of our previously reported multiplex PCR methods for Pectinatus spp. and beer-spoilage cocci were reconstructed. Each modified multiplex PCR method was found specifically to detect beer-spoilage species of Pectinatus and cocci, including new species.     

Sequence of the gene encoding the 16S rRNA of the beer spoilage organismMegasphaera cerevisiae

The 16S ribosomal RNA gene from the beer-spoilage organism,Megasphaera cerevisiae was polymerase chain reaction (PCR)-amplified and sequenced. Analysis confirmed the phylogenetic position ofM. cerevisiae as a sister taxon ofMegasphaera elsdenii, within the obligately anaerobic, Gram-negative cocci. The sequence obtained should facilitate the development of DNA probes for early detection of this spoilage organism.     

Application of Rep-PCR fingerprint in rapid identification of beer-spoilage bacteria

The application potential of rep-PCR in typing beer-spoilage isolates was studied. The effects of different factors, including DNA templates and primers, on the quality and reproducibility of fingerprints were investigated. The CATB protocol was shown to be the feasible method for DNA extraction. Primers BOXA1R and (GTG)₅ were used in rep-PCR, and the PCR products were sequenced to identify strains isolated from two breweries. Rep-PCR fingerprint profiles were obtained using GelCompar II software. Cluster analysis showed that the isolates belonging to Lactobacillus brevis, L. buchneri, L. casei/paracasei, and L. plantarum are divided into 2 or 3 subgroups. In addition, the two rep-PCR fingerprint profiles complemented each other in typing these isolates. By combining the similarity coefficient cut-off (SCC) of species, 9 unknown isolates were rapidly identified using both fingerprint databases. The results indicate that rep-PCR is a simple, reliable, and promising method for the rapid identification of beer-spoilage bacteria.     

Development of Multiplex PCR to Detect Five Pythium Species Related to Turfgrass Diseases

The objective of this study was to develop multiplex PCR detection method for five Pythium species associated with turfgrass diseases, Pythium aphanidermatum, Pythium arrhenomanes, Pythium graminicola, Pythium torulosum and Pythium vanterpoolii. Species‐specific primers and two common primers were designed based on the sequences of the internal transcribed spacer region of ribosomal DNA. Another primer set by which all organisms would be amplified in 18S rDNA was used as a positive control. When these total nine primers were applied to the multiplex PCR, all species were individually discriminated in the mixture of five species culture DNA. Furthermore, all five Pythium species were detected in naturally infected plants using the multiplex PCR.     

Development of a haemolysin gene‐based multiplex PCR for simultaneous detection of Vibrio campbellii,Vibrio harveyi and Vibrio parahaemolyticus

Aim: To develop a haemolysin (hly) gene‐based species‐specific multiplex PCR for simple and rapid detection of Vibrio campbellii, V. harveyi and V. parahaemolyticus. Methods and Results: The complete hly genes of three V. campbellii strains isolated from diseased shrimps were sequenced and species‐specific PCR primers were designed based on these sequences and the registered hly gene sequences of Vibrio harveyi and Vibrio parahaemolyticus. Specificity and sensitivity of the multiplex PCR was validated with 27 V. campbellii, 16 V. harveyi, and 69 V. parahaemolyticus, 18 other Vibrio species, one Photobacterium damselae and nine other bacterial species. The detection limits of all the three target species were in between 10 and 100 cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient to be considered as an effective tool in a prediction system to prevent potential disease outbreak by these Vibrio species. Significance and Impact of the Study: Because there is lack of simple, rapid and cost‐effective method to differentiate these closely related V. campbellii, V. harveyi and V. parahaemolyticus species, the multiplex PCR developed in this study will be very effective in epidemiological, ecological and economical points of view.     

A highly sensitive and specific multiplex PCR assay for simultaneous detection of Vibrio cholerae,Vibrio parahaemolyticus and Vibrio vulnificus

Aims: To develop an effective multiplex PCR for simultaneous and rapid detection of Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus, the three most important Vibrio species that can cause devastating health hazards among human. Methods and Results: Species‐specific PCR primers were designed based on toxR gene for V. cholerae and V. parahaemolyticus, and vvhA gene for V. vulnificus. The multiplex PCR was validated with 488 Vibrio strains including 322 V. cholerae, 12 V. vulnificus, and 82 V. parahaemolyticus, 20 other Vibrio species and 17 other bacterial species associated with human diseases. It could detect the three target bacteria without any ambiguity even among closely related species. It showed good efficiency in detection of co‐existing target species in the same sample. The detection limit of all the target species was ten cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient for simultaneous detection of these potentially pathogenic Vibrio species in clinical and environmental samples. Significance and Impact of the Study: This simple, rapid and cost‐effective method can be applicable in a prediction system to prevent disease outbreak by these Vibrio species and can be considered as an effective tool for both epidemiologist and ecologist.     

Multiplex PCR assay for rapid identification of three endangered snake species of India

Species identification has been the core issue in all approaches of conservation of endangered wild life. In this regard molecular techniques for species authentication have proved indispensable. A novel multiplex PCR assay for the identification of three Indian snake species Python morulus, Ptyas mucosus, and Naja naja is successfully demonstrated using 16S rRNA gene. Three reverse primers and a common forward primer were designed to generate three different size species-specific PCR fragments. Absence of any PCR amplification in non-target species proves the specificity of the primers. These four primers were combined in a multiplex assay to enable identification of three snake species in a single reaction. The assay described here shows its utility in identifying unknown snake specimen and in case of samples yielding low quality DNA. This multiplex PCR technique using novel primers is an unprecedented approach offered for forensic identification of exhibits originating from three Indian snake species. It is expected that this endeavor will help strengthening conservation efforts for these species.     

Novel multiplex polymerase chain reaction primer set for identification of Lactococcus species

Aims: The gram‐positive bacterial genus Lactococcus has been taxonomically classified into seven species (Lactococcus lactis, Lactococcus garvieae, Lactococcus piscium, Lactococcus plantarum, Lactococcus raffinolactis, Lactococcus chungangensis and Lactococcus fujiensis). This study aimed to develop a novel multiplex polymerase chain reaction (PCR) primer set for the identification of the seven lactococcal species, as well as to differentiate the two industrially important dairy subspecies, L. lactis subsp. lactis and L. lactis subsp. cremoris. Methods and Results: A multiplex PCR primer set was designed based on the nucleotide sequences of the 16S rRNA gene of the seven lactococcal species. The specificity of the established one‐step multiplex PCR scheme was verified using more than 200 bacterial strains, in which a complete sequence match was confirmed by partial sequencing of their 16S rRNA gene. Conclusions: The one‐step multiplex PCR enables the identification and speciation of bacterial strains belonging to the genus Lactococcus and the differentiation of strains of L. lactis subsp. lactis and L. lactis subsp. cremoris. Significance and Impact of the Study: This work provides an efficient method for identification of lactococcal strains of industrial importance.     

Development and evaluation of a multiplex PCR for simultaneous detection of five foodborne pathogens

Aims: To develop a rapid multiplex PCR method for simultaneous detection of five major foodborne pathogens (Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, Salmonella Enteritidis and Shigella flexneri, respectively). Methods and Results: Amplification by PCR was optimized to obtain high efficiency. Sensitivity and specificity assays were investigated by testing different strains. With a multipathogen enrichment, multiplex PCR assay was able to simultaneously detect all of the five organisms in artificially contaminated pork samples. The developed method was further applied to retail meat samples, of which 80% were found to be positive for one or more of these five organisms. All the samples were confirmed by traditional culture methods for each individual species. Conclusions: This study reported a rapid multiplex PCR assay using five primers sets for detection of multiple pathogens. Higher consistency was obtained between the results of multiplex PCR and traditional culture methods. Significance and Impact of the Study: This work has developed a reliable, useful and cost‐effective multiplex PCR method. The assay performed equally as well as the traditional cultural method and facilitated the sensitive detection both in artificially contaminated and naturally contaminated samples.     

PCR methods for the rapid detection and identification of four pathogenic Legionella spp. and two Legionella pneumophila subspecies based on the gene amplification of gyrB

A total of 25 gyrB gene sequences from 20 Legionella pneumophila subsp. pneumophila strains and five L. pneumophila subsp. fraseri strains were obtained and analyzed, and a multiplex PCR for the simultaneous detection of Legionella bozemanae, Legionella longbeachae, Legionella micdadei and Legioenella pneumophila, and two single PCRs for the differentiation of L. pneumophila subsp. pneumophila and L. pneumophila subsp. fraseri were established. The multiplex PCR method was shown to be highly specific and reproducible when tested against 41 target strains and 17 strains of other bacteria species. The sensitivity of the multiplex PCR was also analyzed and was shown to detect levels as low as 1 ng of genomic DNA or 10 colony-forming units (CFUs) per milliliter in mock water samples. Sixty-three air conditioner condensed water samples from Shanghai City were examined, and the result was validated using 16S rRNA sequencing. The data reported here demonstrate that the multiplex PCR method described is efficient and convenient for the detection of Legionella species in water samples. Twenty L. pneumophila subsp. pneumophila strains and five L. pneumophila subsp. fraseri strains were used for the validation of the two L. pneumophila subspecies-specific PCR methods, and the results indicated that the two PCR methods were both highly specific and convenient for the identification of L. pneumophila at the subspecies level.     

Development and evaluation of a multiplex real-time PCR assay for the detection and differentiation of Moraxella bovis, Moraxella bovoculi and Moraxella ovis in pure culture isolates and lacrimal swabs collected from conventionally raised cattle

Aim: To develop a multiplex real‐time PCR assay for the detection and differentiation of Moraxella bovis (M. bovis), M. bovoculi and M. ovis. Methods and Results: The multiplex real‐time PCR assay was validated on three reference strains, 57 pure culture isolates and 45 lacrimal swab samples. All reference strains were identified correctly with no cross‐reactions between species. Sequencing of 53 of the 57 culture isolates confirmed the results obtained with the multiplex real‐time PCR, and the assay had 96·5% (55/57) concordance with a Moraxella spp. multiplex conventional PCR assay on the isolates. Among the lacrimal swab samples, the concordance between the multiplex real‐time PCR and culture was 86·7% (39/45) for M. bovoculi and 75·6% (34/45) for M. bovis. Conclusions: The multiplex real‐time PCR assay is specific and sensitive and can be used directly on lacrimal swab samples. Significance and Impact of Study: The lack of a rapid, specific and sensitive detection method is a barrier for determining the roles of M. bovis, M. bovoculi and M. ovis in infectious bovine keratoconjunctivitis cases, and the developed PCR assay will contribute to improved understanding of the epidemiology and pathogenesis of these three Moraxella species.     

Rapid Differentiation and In Situ Detection of 16 Sourdough Lactobacillus Species by Multiplex PCR

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

Detection of European Peristenus digoneutis loan in mirid populations in southern Ontario

Lygus spp. Hahn (Hemiptera: Miridae) are serious pests of a wide variety of economically important crops in North America. A European parasitoid, Peristenus digoneutis Loan (Hymenoptera: Braconidae), released in the northeastern USA for the biological control of Lygus, has successfully established in nine states and in eastern Canada, including southeastern Ontario, southern Quebec and Nova Scotia. To determine the extent to which P. digoneutis has dispersed and established in mirid populations in Ontario, a single-step multiplex PCR assay, designed to differentiate European species P. digoneutis and P. relictus from native North American Peristenus species, was used to identify parasitoid larvae dissected from field-collected Miridae. A total of 222 parasitoid larvae were analysed with the multiplex assay. Most (172) were identified as members of the P. pallipes Curtis complex; however, three from the Niagara region were identified as P. digoneutis. Specimens that did not amplify using the Peristenus multiplex PCR assay were screened with a PCR primer set designed to detect Leiophron (Hymenoptera: Braconidae) species, and most were confirmed to be Leiophron larvae. This study confirms the presence of P. digoneutis species in southern Ontario, and is another example of the utility of molecular methods for the detection of newly introduced or dispersed parasitoids. The presence of exotic P. digoneutis in southern Ontario may expedite future releases to augment already-established populations. Following such releases, the Peristenus multiplex PCR assay will be a useful component of post-release studies to evaluate the success of the biological control programme for Lygus plant bugs in Canada.     

A single-step multiplex PCR assay for the detection of European Peristenus spp., parasitoids of Lygus spp.

Lygus Hahn (Hemiptera: Miridae) are serious pests of agricultural and greenhouse crops throughout North America. In Europe, bivoltine Peristenus Förster (Hymenoptera: Braconidae) species have a significant impact on Lygus populations. Release and establishment of European P. digoneutis Loan in Lygus lineolaris (Palisot de Beauvois) populations in northeastern USA has renewed interest in the intended liberation of European parasitoids for Lygus control in Canada. Accurate identification of natural enemies is the cornerstone of biological control but conventional methods for identifying Peristenus species and estimating parasitism rates rely on tedious and time-consuming dissection and rearing methods. The present study describes species-specific PCR primers for three species of Peristenus, and the use of a multiplex PCR assay to detect P. digoneutis and P. stygicus Loan eggs and larvae from Lygus rugulipennis Poppius nymphs. Results indicate that the primers amplify uniquely sized, species-specific PCR products for the three species and are capable of detecting single eggs in parasitized nymphs within 3 days post-parasitism. Using a multiplex PCR assay, the primers maintain specificity and sensitivity, and allow detection of each of the three species in a single reaction. Although molecular diagnostics have previously been used in the identification of parasitoids and estimation of parasitism rates, this is the first time a single-step multiplex PCR protocol has been described.     

DEVELOPMENT OF A MULTIPLEX PCR ASSAY FOR SIMULTANEOUS DETECTION OF SIX ALEXANDRIUM SPECIES (DINOPHYCEAE)1

In Japan, the bloom seasons of two toxic species, namely, Alexandrium catenella (Whedon et Kof.) Balech and Alexandrium tamiyavanichii Balech, sometimes overlap with those of three nontoxic Alexandrium species, namely, Alexandrium affine (H. Inouye et Fukuyo) Balech, Alexandrium fraterculus (Balech) Balech, and Alexandrium pseudogoniaulax (Biecheler) T. Horig. ex Y. Kita et Fukuyo. In this study, a multiplex PCR assay has been developed that enables simultaneous detection of six Alexandrium species on the basis of differences in the lengths of the PCR products. The accuracy of the multiplex PCR system was assessed using 101 DNA templates of the six target Alexandrium species and 27 DNA templates of 11 nontarget species (128 DNA templates in total). All amplicons obtained from the 101 DNA templates of the target species were appropriately identified, whereas all 27 DNA templates of the nontarget species were not amplified. Species‐specific identification by the multiplex PCR assay was certainly possible from single cells of the target species.     

Rapid identification of the north-western Pacific billfish species using quantitative real-time polymerase chain reaction techniques

Billfishes are important fishery resources traded and consumed worldwide. As morphological traits are usually removed during processing, molecular methods are applied to identify billfish products. In this study, the approaches of quantitative real-time PCR were developed to identify the six billfish species (Istiompax indica, Istiophorus platypterus, Kajikia audax, Makaira nigricans, Tetrapturus angustirostris and Xiphias gladius) widely distributed in the north-western Pacific Ocean. The developed singleplex systems showed high fidelities to each of the six species via either examining the ΔCt values or melting curve patterns. For samples containing multiple species, individual species are identifiable by a quantitative real-time PCR assay that includes all the singleplex systems. A multiplex system was also developed to identify unknown samples composed of a single species. The methods developed in this study provide a fast and high-throughput manner to identify the north-western Pacific billfish species when morphological traits are unavailable, such as in processed products.     

Comparison of PCR-RFLP with 21-plex PCR and rDNA Sequencing for Identification of Clinical Yeast Isolates

Non-albicans Candida species and other rare yeasts have emerged as major opportunistic pathogens in fungal infections. Identification of opportunistic yeasts in developing countries is mainly performed by phenotypic assay, which are time-consuming and prone to errors. The aim of the present study was to evaluate PCR-RFLP as a routinely used identification technique for the most clinically important Candida species in Iran and make a comparison with a novel multiplex PCR, called 21-plex PCR. One hundred and seventy-three yeast isolates from clinical sources were selected and identified with sequence analysis of the D1/D2 domains of rDNA (LSU rDNA) sequencing as the gold standard method. The results were compared with those obtained by PCR-RFLP using MspI restriction enzyme and the 21-plex PCR. PCR-RFLP correctly identified 93.4% of common pathogenic Candida species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and P. kudriavsevii (=?C. krusei)) and was able to identify 45.5% of isolates of the uncommon yeast species compared to the D1/D2 rDNA sequencing. Compared with PCR-RFLP, all common Candida species and 72.7% of uncommon yeast species were correctly identified by the 21-plex PCR. The application of the 21-plex PCR assay as a non-sequence-based molecular method for the identification of common and rare yeasts can reduce turnaround time and costs for the identification of clinically important yeasts and can be applied in resource-limited settings.

     

Multiplex PCR assay for simultaneous detection and differentiation of Mycobacterium tuberculosis, Mycobacterium avium complexes and other Mycobacterial species directly from clinical specimens

Aims: Polymerase chain reaction (PCR) is the most rapid and sensitive method for diagnosing mycobacterial infections and identifying the aetiological Mycobacterial species in order to administer the appropriate therapy and for better patient management. Methods and Results: Two hundred and thirty‐five samples from 145 clinically suspected cases of tuberculosis were processed for the detection of Mycobacterial infections by ZN (Ziehl Neelsen) smear examination, L‐J & BACTEC^TM MGIT‐960 culture and multiplex PCR tests. The multiplex PCR comprised of genus‐specific primers targeting hsp65 gene, Mycobacterium tuberculosis complex‐specific primer targeting cfp10 (Rv3875, esxB) region and Mycobacterium avium complex‐specific primer pairs targeting 16S–23S Internal Transcribed Spacer sequences. The multiplex PCR developed had an analytical sensitivity of 10 fg (3–4 cells) of mycobacterial DNA. The multiplex PCR test showed the highest (77·24%) detection rate, while ZN smear examination had the lowest (20%) detection rate, which was bettered by L‐J culture (34·4%) and BACTEC^TM MGIT‐960 culture (50·34%) methods. The mean isolation time for M. tuberculosis was 19·03 days in L‐J culture and 8·7 days in BACTEC^TM MGIT‐960 culture. Using the multiplex PCR, we could establish M. tuberculosis + M. avium co‐infection in 1·3% HIV‐negative and 2·9% HIV‐positive patients. The multiplex PCR was also highly useful in diagnosing mycobacteraemia in 38·09% HIV‐positive and 15·38% HIV‐negative cases. Conclusions: The developed in‐house multiplex PCR could identify and differentiate the M. tuberculosis and M. avium complexes from other Mycobacterial species directly from clinical specimens. Significance and Impact of the Study: The triplex PCR developed by us could be used to detect and differentiate M. tuberculosis, M. avium and other mycobacteria in a single reaction tube.     

Simultaneous differential detection of human pathogenic and nonpathogenic Vibrio species using a multiplex PCR based on gyrB and pntA genes

Aims: To develop a multiplex PCR targeting the gyrB and pntA genes for Vibrio species differentiation. Methods and Results: Four pairs of primers targeting gyrB gene of Vibrios at genus level and pntA gene of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus were designed. This PCR method precisely identified 250 Vibrio species and demonstrated sensitivity in the range of 4 × 10⁴ CFU ml^?1 (c. 200 CFU per PCR) to 2 × 10³ CFU ml^?1 (c. 10 CFU per PCR). Overall, the gyrB gene marker showed a higher specificity than the dnaJ gene marker for Vibrio detection and was able to distinguish Aeromonas from Vibrio species. Conclusions: The multiplex PCR based on combined gyrB and pntA provides a high discriminatory power in the differentiation between Vibrio alginolyticus and V. parahaemolyticus, and between V. cholerae and Vibrio mimicus. Significance and Impact of the Study: This assay will be useful for rapid differentiation of various Vibrio species from clinical and environmental sources and significantly overcomes the limitations of the conventional methods.