Distribution and diversity of root-lession nematode (Pratylenchus spp.) associated with Miscanthus × giganteus and Panicum virgatum used for biofuels, and species identification in a multiplex polymerase chain reaction

Nematology ◽  
2011 ◽  
Vol 13 (6) ◽  
pp. 673-686 ◽  
Author(s):  
Michael Gray ◽  
Horacio Lopez-Nicora ◽  
Tesfamariam Mekete ◽  
Terry Niblack ◽  
Kimberly Reynolds
Keyword(s):  
Multiplex Pcr ◽  
Panicum Virgatum ◽  
Sequence Data ◽  
Simultaneous Detection ◽  
Morphological Identification ◽  
28S Rrna ◽  
Detection Rates ◽  
The Usa ◽  
Primer Sets ◽  
Species Specific

AbstractThe distribution of Pratylenchus spp. from bioenergy field plots in six states (Illinois, Iowa, South Dakota, Kentucky, Tennessee, Georgia) of the USA were surveyed. The species were identified based on morphology and morphometrics and further characterised based on fragment sequences of the 28S rRNA of the D2-D3 region. The region revealed variations in sequencing information that supports the morphological identification. In this work, six Pratylenchus spp. were detected: Pratylenchus brachyurus, P. crenatus, P. hexincisus, P. neglectus, P. penetrans and P. scribneri. Pratylenchus scribneri, P. crenatus, and P. penetrans were distributed most widely, with detection of 34, 29 and 15%, respectively. Pratylenchus hexincisus, P. brachyurus and P. neglectus were distributed sporadically, with detection rates of 10.0, 2.6 and 2.0%, respectively. A one-step multiplex PCR was developed for the simultaneous detection of P. scribneri, P. crenatus and P. penetrans. Sequence data from this research and NCBI were used to generate different primer sets that are species-specific. We have therefore designed three sets of primers that discriminate P. scribneri, P. crenatus and P. penetrans in multiplex PCR. All the tested primers have shown specificity and have no cross-reaction with the non-target species. When used in a uniplex, duplex and triplex PCR, the selected three primers gave a unique electrophoretic DNA banding pattern characterised by a single DNA fragment for P. scribneri (ca 750), P. crenatus (ca 690), and P. penetrans (ca 520). The method could be used for routine diagnostic programmes.

Simultaneous detection of multiple pathogens by multiplex PCR coupled with DNA biochip hybridization

2017 ◽  
Vol 52 (2) ◽  
pp. 186-195 ◽  
Author(s):  
Hsiang-Yun Tung ◽  
Wei-Chen Chen ◽  
Bor-Rung Ou ◽  
Jan-Ying Yeh ◽  
Yeong-Hsiang Cheng ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Simultaneous Detection ◽  
Clinical Samples ◽  
Single Infection ◽  
Pcr Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Viral Pathogens ◽  
Single Target ◽  
Primer Sets ◽  
Multiple Pathogens

Traditional serological enzyme-linked immunosorbent assay (ELISA) is routinely used to monitor pathogens during quarantine in most animal facilities to prevent possible infection. However, the ELISA platform is a single-target assay, and screening all targeted pathogens is time-consuming and laborious. In this study, to increase sensitivity and to reduce diagnosis time for high-throughput processes, multiplex PCR and DNA biochip techniques were combined to develop a multi-pathogen diagnostic method for use instead of routine ELISA. Eight primer sets were designed for multiplex PCR to detect genes from seven targeted bacterial and viral pathogens. DNA–DNA hybridization was conducted on a biochip following the multiple PCR analysis. Using this method, a total of 24 clinical samples were tested, and the result showed that not only single infection but also co-infection by multi-pathogens can be detected. In conclusion, multiplex PCR coupled with a DNA biochip is an efficient method for detecting multi-pathogens in a reaction. This platform is a useful tool for quarantine services and disease prevention in animal facilities.

A Collagenase-Targeted Multiplex PCR Assay for Identification of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus

2005 ◽  
Vol 68 (1) ◽  
pp. 150-153 ◽  
Author(s):  
ANGELA DI PINTO ◽  
GIUSEPPINA CICCARESE ◽  
GIUSEPPINA TANTILLO ◽  
DOMENICO CATALANO ◽  
VITO TONY FORTE
Keyword(s):  
Multiplex Pcr ◽  
Vibrio Cholerae ◽  
Vibrio Parahaemolyticus ◽  
Food Samples ◽  
Vibrio Alginolyticus ◽  
Pcr Assay ◽  
Molecular Approaches ◽  
Multiplex Pcr Assay ◽  
Primer Sets ◽  
Species Specific

A multiplex PCR assay using three collagenase-targeted primer pairs for the species-specific detection of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus was developed. The results highlight the species specificity of the three primer sets designed. Because of the increasing importance of Vibrio spp. in human foodborne diseases, molecular approaches for routine microbial screening and monitoring of clinical, environmental, and food samples also have become more important. The results of this study indicate that the gene coding for collagenase should be used as an alternative molecular target to discriminate among the three Vibrio species.

Morphological and molecular characterisation of some Hemicriconemoides species (Nematoda: Criconematidae) together with a phylogeny of the genus

Nematology ◽  
2014 ◽  
Vol 16 (5) ◽  
pp. 519-553 ◽  
Author(s):  
Esther Van den Berg ◽  
Louwrens R. Tiedt ◽  
Renato N. Inserra ◽  
Jason D. Stanley ◽  
Nicola Vovlas ◽  
...  
Keyword(s):  
Fruit Trees ◽  
Valid Species ◽  
Rrna Gene ◽  
28S Rrna ◽  
Molecular Characterisation ◽  
Electron Microscopic ◽  
Specific Primers ◽  
Scanning Electron Microscopic ◽  
The Usa ◽  
Species Specific

Sheathoid nematodes of the genusHemicriconemoidesare migratory root-ectoparasites of many plants including various agricultural crops and fruit trees. They are generally found inhabiting warm areas of the world and presently consist of 52 valid species. In this study we provide morphological and molecular characterisation of 12 species of this genusviz.:H. alexis,H. brachyurus,H. californianus,H. chitwoodi,H. macrodorus,H. minutus,H. ortonwilliamsi,H. promissus,H. silvaticus,H. strictathecatus,H. wessoniandHemicriconemoidessp. originating from China, Greece, Japan, Myanmar, Spain, South Africa and the USA. Morphological descriptions, measurements, light and scanning electron microscopic observations and drawings are given for several species. Phylogenetic relationships withinHemicriconemoides, as inferred from the analyses of the D2-D3 of 28S rRNA and ITS-rRNA gene sequences, resulted in trees with three major clades that corresponded with species groupings based on morphology of the lip pattern and vulval flap. PCR with species-specific primers were developed forH. californianus,H. chitwoodiandH. strictathecatus.

Development of a rapid identification method forAeromonasspecies by multiplex-PCR

2005 ◽  
Vol 51 (11) ◽  
pp. 957-966 ◽  
Author(s):  
Keya Sen
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Multiplex Pcr ◽  
Rapid Identification ◽  
Complete Agreement ◽  
Aeromonas Species ◽  
Rrna Gene ◽  
Primer Sets ◽  
Species Specific ◽  
The 16S Rrna Gene

Existing biochemical methods cannot distinguish among some species of Aeromonads, while genetic methods are labor intensive. In this study, primers were developed to three genes of Aeromonas: lipase, elastase, and DNA gyraseB. In addition, six previously described primer sets, five corresponding to species-specific signature regions of the 16S rRNA gene from A. veronii, A. popoffii, A. caviae, A. jandaei, and A. schubertii, respectively, and one corresponding to A. hydrophila specific lipase (hydrolipase), were chosen. The primer sets were combined in a series of multiplex-PCR (mPCR) assays against 38 previously characterized strains. Following PCR, each species was distinguished by the production of a unique combination of amplicons. When the assays were tested using 63 drinking water isolates, there was complete agreement in the species identification (ID) for 59 isolates, with ID established by biochemical assays. Sequencing the gyrB and the 16S rRNA gene from the remaining four strains established that the ID obtained by mPCR was correct for three strains. For only one strain, no consensus ID could be obtained. A rapid and reliable method for identification of different Aeromonas species is proposed that does not require restriction enzyme digestions, thus simplifying and speeding up the process.Key words: Aeromonas, multiplex-PCR, identification.

Species-specific probes for the identification of the African tsetse-transmitted trypanosomes

Parasitology ◽  
2009 ◽  
Vol 136 (12) ◽  
pp. 1501-1507 ◽  
Author(s):  
W. GIBSON
Keyword(s):  
Genetic Diversity ◽  
Phylogenetic Analysis ◽  
Sequence Data ◽  
Quantitative Information ◽  
Morphological Identification ◽  
Trypanosome Species ◽  
Accurate Identification ◽  
Identification Methods ◽  
Detection And Identification ◽  
Species Specific

SUMMARYThe first step in studying the epidemiology of a disease is the accurate identification of the pathogen. Traditional reliance on morphological identification has given way to the use of molecular methods for the detection and identification of pathogens, greatly improving our understanding of epidemiology. For the African tsetse-transmitted trypanosomes, the growth of PCR methods for identification of trypanosomes has led to increased appreciation of trypanosome genetic diversity and discovery of hitherto unknown trypanosome species, as well as greater knowledge about the number and type of trypanosome infections circulating in mammalian hosts and vectors. Sequence data and phylogenetic analysis have provided quantitative information on the relatedness of different trypanosome species and allowed the new trypanosome genotypes discovered through the use of species identification methods in the field to be accurately placed in the phylogenetic tree.

scholarly journals Simultaneous Detection of Pseudomonas fragi, P. lundensis, and P. putida from Meat by Use of a Multiplex PCR Assay Targeting the carA Gene

2007 ◽  
Vol 73 (7) ◽  
pp. 2354-2359 ◽  
Author(s):  
Danilo Ercolini ◽  
Federica Russo ◽  
Giuseppe Blaiotta ◽  
Olimpia Pepe ◽  
Gianluigi Mauriello ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Simultaneous Detection ◽  
Small Subunit ◽  
Carbamoyl Phosphate ◽  
Pcr Assay ◽  
Pseudomonas Fragi ◽  
Multiplex Pcr Assay ◽  
Species Specific ◽  
Simultaneous Identification ◽  
Pork Samples

ABSTRACT Species-specific primers and a multiplex PCR assay were developed for the simultaneous identification and differentiation of Pseudomonas fragi, P. lundensis, and P. putida based on the coamplification of different portions of the small subunit of the carbamoyl phosphate synthase gene (carA). The carA multiplex PCR was used to detect the presence of the three Pseudomonas species from beef, chicken, and pork samples and proved to be effective in showing their evolution during the storage of meat.

scholarly journals Development of a Multiplex PCR-Based Assay for Rapid Serotyping of Erysipelothrix Species

2020 ◽  
Vol 58 (6) ◽  
Author(s):  
Yoshihiro Shimoji ◽  
Kazumasa Shiraiwa ◽  
Haruka Tominaga ◽  
Sayaka Nishikawa ◽  
Masahiro Eguchi ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Simultaneous Detection ◽  
Epidemiological Studies ◽  
Genomic Region ◽  
Erysipelothrix Rhusiopathiae ◽  
Content Type ◽  
Heat Stable ◽  
Wide Range ◽  
Species Specific ◽  
Specific Rabbit

ABSTRACT The Gram-positive bacterium Erysipelothrix rhusiopathiae is a zoonotic pathogen that causes erysipelas in a wide range of mammalian and avian species. Historically, E. rhusiopathiae has been differentiated from other Erysipelothrix species by serotyping. Among 28 serovars of Erysipelothrix species, specific serovars, namely, 1a, 1b, and 2 of E. rhusiopathiae, are associated mainly with the disease in pigs, poultry, and humans; however, other serovar strains are often simultaneously isolated from diseased and healthy animals, indicating the importance of isolate serotyping for epidemiology. The traditional serotyping protocol, which uses heat-stable peptidoglycan antigens and type-specific rabbit antisera in an agar-gel precipitation test, is time-consuming and labor-intensive. To develop a rapid serotyping scheme, we analyzed sequences of the 12- to 22-kb chromosomal region, which corresponds to the genetic region responsible for virulence of serovar 1a and 2 strains of E. rhusiopathiae, of the 28 serovars of Erysipelothrix species. We confirmed that the serovar 13 strain lacks the genomic region and that some serovar strains possess very similar or the same genetic structure, prohibiting differentiation of the serovars. We created 4 multiplex PCR sets allowing the simultaneous detection and differentiation of the majority of Erysipelothrix serovars. Together with a previously reported multiplex PCR that can differentiate serovars 1a, 1b, 2, and 5, the multiplex PCR-based assay developed in this study covers all but one (serovar 13) of the reported serovars of Erysipelothrix species and should be a valuable tool for etiological as well as epidemiological studies of Erysipelothrix infections.

scholarly journals Detection and Differentiation ofListeria spp. by a Single Reaction Based on Multiplex PCR

1999 ◽  
Vol 65 (10) ◽  
pp. 4688-4692 ◽  
Author(s):  
Andreas Bubert ◽  
Inge Hein ◽  
Marcus Rauch ◽  
Angelika Lehner ◽  
ByoungSu Yoon ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Dna Sequences ◽  
Simultaneous Detection ◽  
Specific Gene ◽  
Food Hygiene ◽  
Specific Detection ◽  
Pcr Method ◽  
Species Specific ◽  
Different Sources ◽  
Single Reaction

ABSTRACT The iap gene encodes the protein p60, which is common to all Listeria species. A previous comparison of the DNA sequences indicated conserved and species-specific gene portions. Based on these comparisons, a combination consisting of only five different primers that allows the specific detection and differentiation ofListeria species with a single multiplex PCR and subsequent gel analysis was selected. One primer was derived from the conserved 3′ end and is specific for all Listeria species; the other four primers are specific for Listeria monocytogenes,L. innocua, L. grayi, or the three grouped species L. ivanovii, L. seeligeri, and L. welshimeri, respectively. The PCR method, which also enables the simultaneous detection of L. monocytogenes and L. innocua, was evaluated against conventional biotyping with 200 food hygiene-relevant Listeria strains. The results indicated the superiority of this technique. Thus, this novel type of multiplex PCR may be useful for rapid Listeria species confirmation and for identification of Listeria species for strains isolated from different sources.

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