Quantitative Multiprobe PCR Assay for Simultaneous Detection and Identification to Species Level of Bacterial Pathogens

The prevalence of campylobacter gastroenteritis has been estimated by bacterial isolation using selective culture. However, there is evidence that certain species and strains are not recovered on selective agars. We have therefore compared direct PCR assays of faecal samples with campylobacter culture, and explored the potential of PCR for simultaneous detection and identification to the species level. Two hundred unselected faecal samples from cases of acute gastroenteritis were cultured on modified charcoal cefoperazone deoxycholate agar and subjected to DNA extraction and PCR assay. Culture on CCDA indicated that 16 of the 200 samples contained ‘Campylobacter spp.’. By contrast, PCR assays detected campylobacters in 19 of the 200 samples, including 15 of the culture-positive samples, and further identified them as: C. jejuni (16), C. coli (2) and C. hyointestinalis (1). These results show that PCR offers a different perspective on the incidence and identity of campylobacters in human gastroenteritis.

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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 assay for the simultaneous detection of bacterial pathogens in rainbow trout

Aquaculture International ◽

10.1007/s10499-017-0135-0 ◽

2017 ◽

Vol 25 (4) ◽

pp. 1569-1575 ◽

Cited By ~ 2

Author(s):

Nadia Rajabzadeh ◽

Mohsen Naeemipour ◽

Mohsen Seyedabadi

Keyword(s):

Rainbow Trout ◽

Multiplex Pcr ◽

Bacterial Pathogens ◽

Simultaneous Detection ◽

Pcr Assay ◽

Multiplex Pcr Assay

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Multiplex RT–PCR Assay for the Simultaneous Detection and Identification of Five Sugarcane Viruses

Sugar Tech ◽

10.1007/s12355-020-00805-2 ◽

2020 ◽

Vol 22 (4) ◽

pp. 662-670

Author(s):

Xiao-Yan Feng ◽

Wen-Zhi Wang ◽

Lin-Bo Shen ◽

Jun-Gang Wang ◽

Guo-Ru Xiong ◽

...

Keyword(s):

Simultaneous Detection ◽

Rt Pcr ◽

Pcr Assay ◽

Detection And Identification

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Development of a multiplex real-time PCR assay for the simultaneous detection of four bacterial pathogens causing pneumonia

PLoS ONE ◽

10.1371/journal.pone.0253402 ◽

2021 ◽

Vol 16 (6) ◽

pp. e0253402

Author(s):

Ho Jae Lim ◽

Eun-Rim Kang ◽

Min Young Park ◽

Bo Kyung Kim ◽

Min Jin Kim ◽

...

Keyword(s):

Real Time ◽

Limit Of Detection ◽

Bacterial Pathogens ◽

Simultaneous Detection ◽

Analytical Performance ◽

Patient Treatment ◽

Acid Extraction ◽

Pcr Assay ◽

Microbial Identification ◽

Tract Infections

Classification of clinical symptoms and diagnostic microbiology are essential to effectively employ antimicrobial therapy for lower respiratory tract infections (LRTIs) in a timely manner. Empirical antibiotic treatment without microbial identification hinders the selective use of narrow-spectrum antibiotics and effective patient treatment. Thus, the development of rapid and accurate diagnostic procedures that can be readily adopted by the clinic is necessary to minimize non-essential or excessive use of antibiotics and accelerate patient recovery from LRTI-induced damage. We developed and validated a multiplex real-time polymerase chain reaction (mRT-PCR) assay with good analytical performance and high specificity to simultaneously detect four bacterial pathogens causing pneumonia: Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Moraxella catarrhalis. The analytical performance of mRT-PCR against target pathogens was evaluated by the limit of detection (LOD), specificity, and repeatability. Two hundred and ten clinical specimens from pneumonia patients were processed using an automatic nucleic acid extraction system for the “respiratory bacteria four” (RB4) mRT-PCR assay, and the results were directly compared to references from bacterial culture and/or Sanger sequencing. The RB4 mRT-PCR assay detected all target pathogens from sputum specimens with a coefficient of variation ranging from 0.29 to 1.71 and conservative LOD of DNA corresponding to 5 × 102 copies/reaction. The concordance of the assay with reference-positive specimens was 100%, and additional bacterial infections were detected from reference-negative specimens. Overall, the RB4 mRT-PCR assay showed a more rapid turnaround time and higher performance that those of reference assays. The RB4 mRT-PCR assay is a high-throughput and reliable tool that assists decision-making assessment and outperforms other standard methods. This tool supports patient management by considerably reducing the inappropriate use of antibiotics.

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A novel multiplex PCR assay for simultaneous detection of nine clinically significant bacterial pathogens associated with bovine mastitis

Molecular and Cellular Probes ◽

10.1016/j.mcp.2017.03.004 ◽

2017 ◽

Vol 33 ◽

pp. 57-64 ◽

Cited By ~ 10

Author(s):

Aqeela Ashraf ◽

Muhammad Imran ◽

Tahir Yaqub ◽

Muhammad Tayyab ◽

Wasim Shehzad ◽

...

Keyword(s):

Multiplex Pcr ◽

Bacterial Pathogens ◽

Simultaneous Detection ◽

Bovine Mastitis ◽

Pcr Assay ◽

Multiplex Pcr Assay ◽

Clinically Significant

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Multiplex Polymerase Chain Reaction for Simultaneous Detection of Lawsonia Intracellularis, Serpulina Hyodysen Teriae, and Salmonellae in Porcine Intestinal Specimens

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063879700900309 ◽

1997 ◽

Vol 9 (3) ◽

pp. 281-286 ◽

Cited By ~ 23

Author(s):

Robert O. Elder ◽

Gerald E. Duhamel ◽

Michelle R. Mathiesen ◽

E. Denis Erickson ◽

Connie J. Gebhart ◽

...

Keyword(s):

Dna Sequences ◽

Simultaneous Detection ◽

Lawsonia Intracellularis ◽

Pcr Assay ◽

Swine Dysentery ◽

Bacterial Agent ◽

Detection And Identification ◽

Etiologic Agents ◽

Total Dna ◽

Polymerase Chain

Proliferative enteritis, swine dysentery, and porcine salmonellosis are the most common enteric bacterial diseases affecting pigs in the growing and finishing stages of production. Currently, diagnoses of these diseases by standard cultural techniques of intestinal specimens can be laborious, time consuming, and expensive (swine dysentery, porcine salmonellosis) or impossible (proliferative enteritis). Amplification by polymerase chain reaction (PCR) of DNA sequences specific for each bacterial agent is a highly sensitive and specific method that overcomes the limitations associated with standard detection methods. A multiplex PCR (M-PCR) assay was developed for simultaneous detection and identification of the etiologic agents associated with proliferative enteritis, swine dysentery, and porcine salmonellosis in a single reaction using total DNA obtained directly from intestinal specimens. Purified DNA obtained from pure cultures of each bacterial agent alone or mixed in different combinations and concentrations and total DNA from intestinal specimens were amplified using the Lawsonia intracellularis-, Serpulina hyodysenteriae-, and salmonellae-specific M-PCR assay. Intestinal specimens consisted of feces and mucosal scrapings obtained from field cases of each disease alone or in combinations and feces obtained from pigs challenged with S. hyodysenteriae. The banding pattern of the amplified PCR products, after agarose gel electrophoresis and staining, indicated the presence of individual or combinations of etiologic agents in each specimen. Results from this study indicated that simultaneous amplification of L. intracellularis-, S. hyodysenteriae-, and salmonellae-specific DNA sequences by M-PCR can be used for specific detection and identification of three major enteric bacterial pathogens associated with proliferative enteritis, swine dysentery, and porcine salmonellosis occurring alone or in combinations. Also, the M-PCR assay can be done using DNA obtained directly from intestinal specimens submitted for diagnostic investigation.

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