Development of multiplex PCR assay for concurrent detection of tick borne haemoparasitic infections in bovines

Abstract This study describes development and evaluation of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bigemina and Anaplasma marginale infections in bovines. The assay was developed using parasites specific genomic DNA and three sets of PCR primers targeting the Tams1, 18S rRNA and 16S rRNA genes of T. annulata, B. bigemina and A. marginale, respectively. Blood samples collected from a total of 461 bovines, suspected for haemoparasitic infections, were examined microscopically to record the status of infection and simultaneously, genomic DNA extracted from these blood samples were utilized for the optimization and validation of multiplex PCR assay. Microscopic examination of blood samples revealed presence of single and multiple species of haemoparasites in 25.8% and 2.4% samples, respectively. Results of multiplex PCR revealed the presence of single haemoparasitic species infection in 159 cases (34.5%), whereas mixed infection was recorded in 82 (17.8%) samples. Occurrence of individual species infection detected by mPCR in the study was 26.03% (120/461) for T. annulata, 3.25% (15/461) for B. bigemina and 5.20% (24/461) for A. marginale. The detection limit of multiplex PCR assay was at the template dilutions of 10−6, 10−6 and 10−4, which corresponded to 0.1 pg, 0.1 pg and 10.0 pg of DNA for T. annulata, A. marginale, and B. bigemina, respectively. Based on the high diagnostic sensitivity and throughput, multiplex PCR assay developed in the present study could be exploited as a tool to conduct large-scale epidemiological survey for tick-borne haemoparasitic infection of bovines.

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Simultaneous Detection of Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri, Three Major Fish Pathogens, by Multiplex PCR

Applied and Environmental Microbiology ◽

10.1128/aem.68.10.5177-5180.2002 ◽

2002 ◽

Vol 68 (10) ◽

pp. 5177-5180 ◽

Cited By ~ 70

Author(s):

A. del Cerro ◽

I. Marquez ◽

J. A. Guijarro

Keyword(s):

Multiplex Pcr ◽

Simultaneous Detection ◽

Aeromonas Salmonicida ◽

16S Rrna Genes ◽

Rrna Genes ◽

Yersinia Ruckeri ◽

Pcr Assay ◽

Fish Pathogens ◽

Flavobacterium Psychrophilum ◽

Multiplex Pcr Assay

ABSTRACT A multiplex PCR assay based on the 16S rRNA genes was developed for the simultaneous detection of three major fish pathogens, Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri. The assay proved to be specific and as sensitive as each single PCR assay, with detection limits in the range of 6, 0.6, and 27 CFU for A. salmonicida, F. psychrophilum, and Y. ruckeri, respectively. The assay was useful for the detection of the bacteria in artificially infected fish as well as in fish farm outbreaks. Results revealed that this multiplex PCR system permits a specific, sensitive, reproducible, and rapid method for the routine laboratory diagnosis of infections produced by these three bacteria.

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A PCR method targeting internal transcribed spacers: the simultaneous detection of Babesia bigemina and Babesia bovis in cattle

Acta Parasitologica ◽

10.2478/s11686-014-0222-6 ◽

2014 ◽

Vol 59 (1) ◽

Cited By ~ 8

Author(s):

Junlong Liu ◽

Guiquan Guan ◽

Aihong Liu ◽

Youquan Li ◽

Hong Yin ◽

...

Keyword(s):

Light Microscopy ◽

Multiplex Pcr ◽

Simultaneous Detection ◽

Internal Transcribed Spacers ◽

Babesia Bovis ◽

Babesia Bigemina ◽

Pcr Assay ◽

Blood Samples ◽

Multiplex Pcr Assay ◽

Pcr Method

AbstractIn this study, two pairs of oligonucleotide primers were designed according to the nucleotide sequence of the internal transcribed spacers (ITSs) of Babesia bigemina and B. bovis isolates from China. The primers were used in a multiplex PCR to detect parasite DNA in blood samples from cattle. There was no cross reactions with B. ovata, B. major, B. sp. Kashi, Theileria annulata, T. sergenti, T. sinensis or normal bovine DNA. The sensitivity of multiplex PCR assay was 1 pg and 10 pg DNA for B. bigemina and B. bovis, respectively. A total of 260 field blood samples collected from cattle in five provinces of China were analyzed by multiplex PCR and light microscopy. PCR testing revealed that 7.3% (19/260) and 5.8% (15/260) of cattle were positive for B. bigemina and B. bovis and 1.2% (3/260) of cattle were co-infected with B. bigemina and B. bovis. Using light microscopy, 2.3% (6/260) and 1.5% (4/260) of cattle were infected by B. bigemina and B. bovis, respectively, and no co-infection was found. The results showed that the multiplex PCR developed in the present study could be an alternative diagnostic tool for the detection of B. bovis and B. bigemina infection in cattle.

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Multiplex PCR assay for ureC and 16S rRNA genes clearly discriminates between both subspecies of Photobacterium damselae

Diseases of Aquatic Organisms ◽

10.3354/dao040177 ◽

2000 ◽

Vol 40 ◽

pp. 177-183 ◽

Cited By ~ 53

Author(s):

CR Osorio ◽

AE Toranzo ◽

JL Romalde ◽

JL Barja

Keyword(s):

16S Rrna ◽

Multiplex Pcr ◽

16S Rrna Genes ◽

Rrna Genes ◽

Pcr Assay ◽

Multiplex Pcr Assay ◽

Photobacterium Damselae

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Development and evaluation of a multiplex PCR assay for simultaneous detection ofFlavobacterium psychrophilum,Yersinia ruckeriandAeromonas salmonicidasubsp.salmonicidain culture fisheries

Journal of Veterinary Science ◽

10.4142/jvs.2010.11.3.235 ◽

2010 ◽

Vol 11 (3) ◽

pp. 235 ◽

Cited By ~ 6

Author(s):

Ertan Emek Onuk ◽

Alper Ciftci ◽

Arzu Findik ◽

Yuksel Durmaz

Keyword(s):

Multiplex Pcr ◽

Simultaneous Detection ◽

Pcr Assay ◽

Multiplex Pcr Assay

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A new one-step multiplex PCR assay for simultaneous detection and identification of avian haemosporidian parasites

Parasitology Research ◽

10.1007/s00436-018-6153-7 ◽

2018 ◽

Vol 118 (1) ◽

pp. 191-201 ◽

Cited By ~ 17

Author(s):

Arif Ciloglu ◽

Vincenzo A. Ellis ◽

Rasa Bernotienė ◽

Gediminas Valkiūnas ◽

Staffan Bensch

Keyword(s):

Multiplex Pcr ◽

Simultaneous Detection ◽

Pcr Assay ◽

Haemosporidian Parasites ◽

Multiplex Pcr Assay ◽

Detection And Identification ◽

One Step

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Multiplex PCR Detection of Enterotoxin Genes in Aeromonas spp. from Suspect Food Samples in Northern Taiwan

Journal of Food Protection ◽

10.4315/0362-028x-71.10.2094 ◽

2008 ◽

Vol 71 (10) ◽

pp. 2094-2099 ◽

Cited By ~ 14

Author(s):

YU-CHANG CHANG ◽

JAN-YI WANG ◽

AMMAIYAPPAN SELVAM ◽

SHU-CHEN KAO ◽

SHANG-SHYNG YANG ◽

...

Keyword(s):

Multiplex Pcr ◽

Diarrheal Disease ◽

Simultaneous Detection ◽

Food Poisoning ◽

Food Samples ◽

Pcr Assay ◽

Multiplex Pcr Assay ◽

Enterotoxin Genes ◽

Causative Agents ◽

Northern Taiwan

Aeromonads possess an array of virulence factors and are causative agents of a number of human infections. Among them, genes of one cytotoxic (Act) and two cytotonic (Alt, Ast) enterotoxins are implicated in a human diarrheal disease. A rapid, specific, simultaneous detection of these enterotoxin genes in suspected food poisoning samples is not yet reported. Hence, a multiplex PCR assay was designed to amplify the cytotoxic (act), heat-labile cytotonic (alt), and heat-stable cytotonic (ast) enterotoxin genes of aeromonads. The PCR assay was tested with 133 Aeromonas spp. isolated from suspect food poisoning samples and retail samples of poultry and fish from wet markets in and around Taipei, Northern Taiwan. The Aeromonas spp. isolates were divided into six genotypes based on absence or presence of one or more enterotoxin genes. Of these 133 isolates, Aeromonas caviae (52.5%) and Aeromonas hydrophila (43.4%) were the most frequently isolated species from food poisoning samples and retail samples, respectively. Among the species, A. hydrophila had a significantly higher proportion for harboring three enterotoxin genes than had the others, whereas Aeromonas encheleia, considered a nonpathogen, was found harboring three enterotoxin genes. The multiplex PCR assays are rapid and specific, and provide a useful tool for the detection and genotyping of enterotoxin genes of aeromonads.

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