A Thermostabilized, One-Step PCR Assay for Simultaneous Detection ofKlebsiella pneumoniaeandHaemophilus influenzae

Klebsiella pneumoniaeandHaemophilus influenzaeare two common pathogens associated with respiratory tract infections. The identification of these pathogens using conventional molecular diagnostic tests requires trained personnel, cold-chain transportation, and storage-dependance, which does not render them user-friendly. The aim of this study was to develop a thermostabilized, cold-chain-free, one-step multiplex PCR for simultaneous detection ofK. pneumoniaeandH. influenzae.The multiplex PCR assay was designed to amplify thephpgene ofK. pneumoniae(202 bp) andp6gene ofH. influenzae(582 bp). In addition, the specific primer to amplifyglmgene ofHelicobacter pylori(105 bp) was included as an internal amplification control. Subsequently, the designed primers and all PCR reagents were thermostabilized by lyophilization. The stability of the thermostabilized PCR was evaluated using theQ10method. The sensitivity and specificity of performances for thermostabilized PCR were evaluated using 127 clinical isolates and were found to be 100% sensitive and specific. The thermostabilized PCR mix was found to be stable for 30 days and theQ10 accelerated stability was found to be 3.02 months. A cold-chain-free, PCR assay for easy, rapid, and simultaneous detection ofK. pneumoniaeandH. influenzaewas successfully developed in this study.

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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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Epidemiological analysis and rapid detection by one-step multiplex PCR assay of Haemophilus influenzae in children with respiratory tract infections in Zhejiang Province, China

BMC Infectious Diseases ◽

10.1186/s12879-018-3295-2 ◽

2018 ◽

Vol 18 (1) ◽

Author(s):

Xingli Fan ◽

Xiaoxiang Liu ◽

Lei Ji ◽

Damin Cai ◽

Jinqin Jiang ◽

...

Keyword(s):

Respiratory Tract ◽

Haemophilus Influenzae ◽

Multiplex Pcr ◽

Rapid Detection ◽

Respiratory Tract Infections ◽

Pcr Assay ◽

Epidemiological Analysis ◽

Multiplex Pcr Assay ◽

One Step ◽

Tract Infections

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A multiplex PCR assay for the simultaneous detection of Taenia hydatigena, T. multiceps, T. pisiformis, and Dipylidium caninum infections

BMC Infectious Diseases ◽

10.1186/s12879-019-4512-3 ◽

2019 ◽

Vol 19 (1) ◽

Author(s):

Guo-Qiang Zhu ◽

Li Li ◽

John Asekhaen Ohiolei ◽

Yan-Tao Wu ◽

Wen-Hui Li ◽

...

Keyword(s):

Multiplex Pcr ◽

Simultaneous Detection ◽

Reaction System ◽

Control Program ◽

Molecular Diagnostic ◽

Pcr Assay ◽

Taenia Hydatigena ◽

Dipylidium Caninum ◽

Multiplex Pcr Assay ◽

Field Samples

Abstract Background Taenia hydatigena, T. multiceps, T. pisiformis, and Dipylidium caninum are four common large and medium-sized tapeworms parasitizing the small intestine of dogs and other canids. These parasites cause serious impact on the health and development of livestock. However, there are, so far, no commercially available molecular diagnostic kits capable of simultaneously detecting all four parasites in dogs. The aim of the study was therefore to develop a multiplex PCR assay that will accurately detect all four cestode infections in one reaction. Methods Specific primers for a multiplex PCR were designed based on corresponding mitochondrial genome sequences, and its detection limit was assessed by serial dilutions of the genomic DNAs of tapeworms examined. Furthermore, field samples of dog feces were tested using the developed assay. Results A multiplex polymerase chain reaction (PCR) assay was developed based on mitochondrial DNA (mtDNA) that accurately and simultaneously identify four cestode species in one reaction using specific fragment sizes of 592, 385, 283, and 190 bp for T. hydatigena, T. multiceps, T. pisiformis, and D. caninum, respectively. The lowest DNA concentration detected was 1 ng for T. hydatigena, T. multiceps and T. pisiformis, and 0.1 ng for D. caninum in a 25 μl reaction system. This assay offers high potential for the rapid detection of these four tapeworms in host feces simultaneously. Conclusions This study provides an efficient tool for the simultaneous detection of T. hydatigena, T. multiceps, T. pisiformis, and D. caninum. The assay will be potentially useful in epidemiological studies, diagnosis, and treatment of these four cestodes infections during prevention and control program.

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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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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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