scholarly journals Development of a Multiplex PCR Assay for Genotyping the Fish Pathogen Piscirickettsia salmonis Through Comparative Genomics

2021 ◽  
Vol 12 ◽  
Author(s):  
Adolfo Isla ◽  
J. Eduardo Martinez-Hernandez ◽  
Héctor A. Levipan ◽  
Denise Haussmann ◽  
Jaime Figueroa ◽  
...  
Keyword(s):  
Comparative Genomics ◽  
Multiplex Pcr ◽  
Core Genome ◽  
Bacterial Pathogen ◽  
Economic Losses ◽  
Coding Sequences ◽  
Multiplex Pcr Assay ◽  
Field Samples ◽  
Pcr Products ◽  
Piscirickettsia Salmonis

Piscirickettsia salmonis is a bacterial pathogen that severely impact the aquaculture in several countries as Canada, Scotland, Ireland, Norway, and Chile. It provokes Piscirickettsiosis outbreaks in the marine phase of salmonid farming, resulting in economic losses. The monophyletic genogroup LF-89 and a divergent genogroup EM-90 are responsible for the most severe Piscirickettsiosis outbreaks in Chile. Therefore, the development of methods for quick genotyping of P. salmonis genogroups in field samples is vital for veterinary diagnoses and understanding the population structure of this pathogen. The present study reports the development of a multiplex PCR for genotyping LF-89 and EM-90 genogroups based on comparative genomics of 73 fully sequenced P. salmonis genomes. The results revealed 2,322 sequences shared between 35 LF-89 genomes, 2,280 sequences in the core-genome of 38 EM-90 genomes, and 331 and 534 accessory coding sequences each genogroup, respectively. A total of 1,801 clusters of coding sequences were shared among all tested genomes of P. salmonis (LF-89 and EM-90), with 253 and 291 unique sequences for LF-89 and EM-90 genogroups, respectively. The Multiplex-1 prototype was chosen for reliable genotyping because of differences in annealing temperatures and respective reaction efficiencies. This method also identified the pathogen in field samples infected with LF-89 or EM-90 strains, which is not possible with other methods currently available. Finally, the genome-based multiplex PCR protocol presented in this study is a rapid and affordable alternative to classical sequencing of PCR products and analyzing the length of restriction fragment polymorphisms.

scholarly journals Multiplex PCR Assay for Detection of Vibrio vulnificus Biotype 2 and Simultaneous Discrimination of Serovar E Strains

2007 ◽  
Vol 73 (6) ◽  
pp. 2029-2032 ◽  
Author(s):  
Eva Sanjuán ◽  
Carmen Amaro
Keyword(s):  
Multiplex Pcr ◽  
Vibrio Vulnificus ◽  
Simultaneous Discrimination ◽  
Fish Pathogen ◽  
Pcr Assay ◽  
Culture Collections ◽  
Multiplex Pcr Assay ◽  
Detection And Identification ◽  
Field Samples ◽  
Zoonotic Strains

ABSTRACT In the present work we develop a multiplex PCR assay for the detection and identification of the fish pathogen Vibrio vulnificus biotype 2 with discriminating potential for zoonotic strains (serovar E). The PCR assay allowed the identification of two new biotype 2 serovar E human isolates from culture collections. Finally, the multiplex was successfully applied to both diagnosis and carrier detection in field samples.

Development of multiplex PCR assay for simultaneous detection of five cucumber pathogens based on comparative genomics

2019 ◽  
Vol 48 (4) ◽  
pp. 369-372
Author(s):  
Jia Yu ◽  
Yuanyuan Zhao ◽  
Gan Ai ◽  
Heng Xu ◽  
Daolong Dou ◽  
...  
Keyword(s):  
Comparative Genomics ◽  
Multiplex Pcr ◽  
Simultaneous Detection ◽  
Pcr Assay ◽  
Multiplex Pcr Assay

scholarly journals Detection of Nocardia, Streptomyces and Rhodococcus from bronchoalveolar lavage specimens of patients with HIV by Multiplex PCR Assay

1970 ◽  
Vol 29 (6) ◽  
Author(s):  
Hossein Ali Rahdar ◽  
Mohammad Reza Salehi ◽  
Abass Bahador ◽  
Seyedesomaye Jasemi ◽  
Morteza Karami-Zarandi ◽  
...  
Keyword(s):  
Bronchoalveolar Lavage ◽  
Multiplex Pcr ◽  
Target Genes ◽  
Pcr Assay ◽  
Accurate Identification ◽  
Streptomyces Spp ◽  
Multiplex Pcr Assay ◽  
Pcr Products ◽  
Pure Cultures ◽  
Immune Health

Background: Nocardia, Streptomyces and Rhodococcus are life threatening opportunistic pathogens under immunodeficiency conditions, particularly among patients infected with HIV. Rapid and accurate detection of these infections can improve immune health quality, patient management and appropriate treatment. The aim of this study was to design a novel multiplex-PCR assay for rapid diagnosis of these three organisms directly from bronchoalveolar lavage (BAL) specimens of patients infected with HIV.Methods: The genus specific primers were designed for directdetection of Nocardia, Streptomyces and Rhodococcus in a single tube multiplex PCR. This PCR specifically amplified the target genes from pure cultures. It subsequently was applied on BAL specimens of 29 HIV positive patients that had previously been culture negative for actinomycete bacteria, of which Nocardia, Streptomyces and Rhodococcus are members.Results: Of 29 respiratory clinical specimens, there were positive for Nocardia spp. and one was positive for Streptomyces spp using the multiplex PCR assay. The sequencing of the PCR products identified the species as Nocardia cyriacigeorgica (n=2), Nocardia farcinica and Streptomyces albus.Conclusion: This novel multiplex PCR assay yielded reliable results for accurate identification of Nocardia, Streptomyces and Rhodococcus from BAL while the results of bacterial culture were negative. 

scholarly journals Development of a multiplex PCR assay for identification of Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis

2007 ◽  
Vol 56 (11) ◽  
pp. 1467-1473 ◽  
Author(s):  
Wataru Yamazaki-Matsune ◽  
Masumi Taguchi ◽  
Kazuko Seto ◽  
Ryuji Kawahara ◽  
Kentaro Kawatsu ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Campylobacter Jejuni ◽  
Campylobacter Coli ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Campylobacter Fetus ◽  
Multiplex Pcr Assay ◽  
Pcr Products ◽  
Campylobacter Lari ◽  
Campylobacter Upsaliensis

A multiplex PCR assay has been developed for the identification of the six common Campylobacter taxa associated with human gastroenteritis and/or septicaemia, namely Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis. The assay was developed using a combination of newly designed and published primers. It provided a specific PCR product for each of the five Campylobacter species and the one subspecies, and each of the PCR products was sufficiently distinguished by a difference in size by agarose gel electrophoresis. On evaluation of efficacy with 142 Campylobacter strains, the assay correctly identified all strains as 1 of the 6 Campylobacter taxa. This multiplex PCR assay is a rapid, simple and practical tool for identification of the six Campylobacter taxa commonly associated with gastroenteritis and/or septicaemia in humans, and offers an effective alternative to conventional biochemical-based assays.

scholarly journals Establishment of a Multiplex – PCR protocol for detection of Y chromosome microdeletion in Azoospermia male patients

2015 ◽  
Vol 18 (4) ◽  
pp. 5-15
Author(s):  
Phuong Thi Kim Huynh ◽  
Thanh Kieu Huynh ◽  
Nam Tri Vo ◽  
Anh Le Tuan Nguyen ◽  
Hoang Duc Nguyen ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Y Chromosome ◽  
Control Sample ◽  
Current Approach ◽  
Marker Genes ◽  
Dna Electrophoresis ◽  
Azoospermia Factor ◽  
Multiplex Pcr Assay ◽  
Pcr Products ◽  
Male Patients

Microdeletion on the Y chromosome is one of the causes that makes men infertile, accounting for 2-10 % of all infertility cases, and occurs frequently at 3 regions of the Ychromosome long arm namely AZFa, AZFb and AZFc (azoospermia factor). Currently, the diagnosis of microdeletion on the Y chromosome is almost mandatory in institutes and centers for infertility diseases before selecting treatment or assisting methods. To detect microdeletion in AZF, SRY and ZFY regions, the current approach is a Multiplex – PCR assay offering by European Academy of Andrology/European Molecular Genetics Quality Network (EAA/ EMQN). However, the drawback of this method is the PCR products posess similar size and then the DNA electrophoresis bands were very close on gels causing the difficult in diagnosis. Therefore, in this study, we have redesigned primer pairs matching with genes that were recommended by EAA/EMQN but the PCR products are clearly different in sizes, making the DNA electrophoresis bands take apart further to facilitate the diagnosis. Besides, we have also created recombinant plasmids carrying the marker genes for the control sample in kits.

Molecular typing of Streptococcus agalactiae isolates of serotype Ia from tilapia in southern China

2019 ◽  
Vol 366 (13) ◽  
Author(s):  
Youlu Su ◽  
Chan Liu ◽  
Yiqin Deng ◽  
Changhong Cheng ◽  
Hongling Ma ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Streptococcus Agalactiae ◽  
Virulence Genes ◽  
Southern China ◽  
Virulence Gene ◽  
Economic Losses ◽  
Putative Virulence Gene ◽  
Gene Profiles ◽  
Multiplex Pcr Assay

ABSTRACT Streptococcus agalactiae is an important pathogen of tilapia causing enormous economic losses worldwide. In this study, multilocus sequence typing indicated that 75 S. agalactiae isolates from tilapia in southern China belonged to sequence type-7, as well as belonging to serotype Ia, as confirmed by multiplex PCR assay. The putative-virulence gene profiles and genetic variation of these strains were determined by three sets of multiplex PCR and multi-virulence locus sequencing typing (MVLST), respectively. Analysis of putative-virulence gene profiles showed that each strain harbored 18 putative-virulence genes but lacked lmb and scpB. Three putative-virulence genes (srr-1, bibA and fbsA) were further selected for MVLST analysis. Our data showed that the strains had 14 MVLST types (1–14) and clustered in three groups (Groups I–Ⅲ). The period of time during 2013 and 2014 was an important turning point for the differentiation of the putative-virulence genes of S. agalactiae, as type 1 within Group Ⅱ became the predominant MVLST type. There were significant differences in MVLST types of S. agalactiae isolated from different tilapia farming regions. MVLST assay may improve the discriminatory power and is suitable for understanding the epidemiology of S. agalactiae serotype Ia and screening multivalent vaccine candidate strains.

scholarly journals Development of Freeze-dried Reagents based Multiplex PCR Assay for the Detection of Common and Emerging Abortion-causing Pathogens

2021 ◽  
Author(s):  
Pallavi Deol ◽  
Sukdeb Nandi ◽  
Vishal Chander ◽  
Chandan Prakash ◽  
Sonalika Mahajan ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Simultaneous Detection ◽  
Economic Losses ◽  
Pcr Assay ◽  
Reliable Technique ◽  
Diarrhea Virus ◽  
Multiplex Pcr Assay ◽  
Freeze Dried ◽  
Pcr Multiplex ◽  
Trained Manpower

Bovine abortion is economically one of the most devastating problems faced by dairy farmers. Apart from non-infectious causes, several infectious pathogens are responsible for abortions, which sometimes manifests as abortion storms. Vaccine against several pathogens is available, in spite of that, abortions cause huge economic losses for the dairy sector. Timely and accurate identification of the etiological agent helps in adopting the mitigation steps to control the damage caused. In addition to the common abortion-causing pathogens such as Brucella abortus, Bovine herpesvirus-1 (BHV-1), bovine viral diarrhea virus (BVDV), several emerging viral causes are being investigated for their possible role in abortion, either exclusively or as co-infection. Molecular methods are widely accepted for the identification of the involved pathogens. However, these assays require individual screening against each pathogen which is time-consuming and uneconomical, hence the multiplex format of PCR assays has been adopted by several laboratories. Multiplexing in real-time PCR is a sensitive and reliable technique, but it requires trained manpower and sophisticated equipment which is largely unavailable in regional disease diagnostic laboratories in India. Hence, in this study, a user-friendly, ready-to-use, gel-based RT-PCR multiplex assay was developed for simultaneous detection of three common pathogens (B. abortus, BHV-1, and BVDV) and two emerging pathogens; bluetongue virus (BTV) as a cause of abortions in bovine and Schmallenberg virus (SBV). After the standardization of the assay, a panel of 211 samples was screened. A high degree of concordance was observed which indicates the developed multiplex PCR assay is reliable and has the potential for screening at regional diagnostic laboratories.

scholarly journals Development of an indirect ELISA to detect Corynebacterium pseudotuberculosis specific antibodies in sheep employing T1 strain culture supernatant as antigen

2013 ◽  
Vol 33 (11) ◽  
pp. 1296-1302 ◽  
Author(s):  
Miriam F. Rebouças ◽  
Dan Loureiro ◽  
Bruno L. Bastos ◽  
Lilia F. Moura-Costa ◽  
Samira A. Hanna ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Indirect Elisa ◽  
Brain Heart Infusion ◽  
Etiologic Agent ◽  
Corynebacterium Pseudotuberculosis ◽  
Broth Culture ◽  
Economic Losses ◽  
Ifn Gamma ◽  
Multiplex Pcr Assay ◽  
Two Samples

Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis (CLA), a chronic disease that affects goats and sheep, characterized by granuloma formation in subcutaneous and internal lymph nodes. CLA causes significant economic losses to commercial goat herds. In this study, we aimed to test secreted antigens secreted from T1 strain bacteria grown in brain heart infusion (BHI) broth in an indirect ELISA system to determine the presence of specific immunoglobulins against C. pseudotuberculosis. We analyzed the BHI antigen electrophoretic profile and the recognition pattern by infected sheep sera samples. The ELISA results were compared with multiplex PCR assay and IFN-gamma production. The ELISA was able to discriminate between negative and positive animals, with a sensitivity of 89% and a specificity of 99%, using microbiological isolation as gold standard. When this assay was compared with multiplex PCR and specific IFN-gamma quantification, six discrepant results were found among thirty-two samples. We concluded that the ELISA using antigens secreted from C. pseudotuberculosis T1 strain growth in BHI broth culture can be used for the serodiagnosis of CLA in sheep.

scholarly journals Comparative Genomics to Develop a Specific Multiplex PCR Assay for Detection of Clavibacter michiganensis

2020 ◽  
Vol 110 (3) ◽  
pp. 556-566
Author(s):  
Shree P. Thapa ◽  
Michael O’Leary ◽  
Marie-Agnès Jacques ◽  
Robert L. Gilbertson ◽  
Gitta Coaker
Keyword(s):  
Comparative Genomics ◽  
Multiplex Pcr ◽  
Internal Control ◽  
Management Strategies ◽  
Detection Methods ◽  
Clavibacter Michiganensis ◽  
Pcr Assay ◽  
Tomato Seed ◽  
Plant Materials ◽  
Multiplex Pcr Assay

Clavibacter michiganensis is a Gram-positive bacterial pathogen that proliferates in the xylem vessels of tomato, causing bacterial wilt and canker symptoms. Accurate detection is a crucial step in confirming outbreaks of bacterial canker and developing management strategies. A major problem with existing detection methods are false-positive and -negative results. Here, we report the use of comparative genomics of 37 diverse Clavibacter strains, including 21 strains sequenced in this study, to identify specific sequences that are C. michiganensis detection targets. Genome-wide phylogenic analyses revealed additional diversity within the genus Clavibacter. Pathogenic C. michiganensis strains varied in plasmid composition, highlighting the need for detection methods based on chromosomal targets. We utilized sequences of C. michiganensis-specific loci to develop a multiplex PCR-based diagnostic platform using two C. michiganensis chromosomal genes (rhuM and tomA) and an internal control amplifying both bacterial and plant DNA (16s ribosomal RNA). The multiplex PCR assay specifically detected C. michiganensis strains from a panel of 110 additional bacteria, including other Clavibacter spp. and bacterial pathogens of tomato. The assay was adapted to detect the presence of C. michiganensis in seed and tomato plant materials with high sensitivity and specificity. In conclusion, the described method represents a robust, specific tool for detection of C. michiganensis in tomato seed and infected plants.

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