Rapid and Sensitive Detection of Major Uropathogens in a Single-Pot Multiplex PCR Assay

Abstract

Urinary tract infection (UTI) is among the most common bacterial infections and poses a significant healthcare burden. Escherichia coli is the most common cause of UTI accounting for up to 70 % and a variable contribution from Proteus mirabilis, Pseudomonas aeruginosa and Klebsiella pneumoniae. To establish a complete diagnostic system, we have developed a single-tube multiplex PCR assay (mPCR) for the detection of the above-mentioned four major uropathogens. The sensitivity of the assay was found to be as low as 10(2) cfu/ml of cells. The mPCR evaluated on 280 clinical isolates detected 100 % of E. coli, P. aeruginosa, P. mirabilis and 95 % of K. pneumonia. The assay was performed on 50 urine samples and found to be specific and sensitive for clinical diagnosis. In addition, the mPCR was also validated on spiked urine samples using 40 clinical isolates to demonstrate its application under different strain used in this assay. In total, mPCR reported here is a rapid and simple screening tool that can compete with conventional biochemical-based screening assays that may require 2-3 days for detection.

Evidence of Sharing of Klebsiella pneumoniae Strains between Healthy Companion Animals and Cohabiting Humans

“…Up to five K. pneumoniae suspected colonies per participant were isolated and stored in 20% glycerol (Sigma-Aldrich, USA) brain heart infusion broth (Biokar Diagnostics, France) at Ϫ20°C until processing. Total DNA was extracted by the boiling method and K. pneumoniae species confirmed by PCR as previously described (19,20).…”

Modern clinical microbiology: new challenges and solutions

“…RT-PCR assays can target either specific pathogens and resistance-or virulence-encoding genes, or universal targets, such as 16S rDNA for bacteria and rDNA internal transcribed spacers for fungi. In addition, RT-PCR multiplex assays can simultaneously target several DNA fragments, thus enabling the detection of a whole panel of pathogens that are implicated in particular syndromes, such as bloodstream infections 83 , communityacquired pneumonia, meningitis 84 , sexually transmitted diseases 85 or urinary tract infections 86 .…”

Implications of direct amplification for measuring antimicrobial resistance using point-of-care devices

“…The use of LAMP for direct detection of pathogens in urine has also been demonstrated targeting the malB gene specific to E. coli . 51 Though PCR is typically inhibitory at urea concentrations larger than 50 mM, 52 LAMP in this study was not inhibited. Direct amplification of targets from swab has been reported with enterovirus, 53 with results in concordance of 86.8% and 100% sensitivity and specificity compared to DNA extraction and PCR.…”

“…62 In urine, PCR is more notably inhibited by urea at concentrations greater than 50 mM. 52 A direct PCR method originally developed for plants has also been used with swabs of cheeks and skin, where clinical swabs are directly added into wells containing the buffer solution. 74 Some direct PCR-based approaches are also available commercially as part of sample collection devices ( e.g .…”