Rapid and Sensitive Detection of Major Uropathogens in a Single-Pot Multiplex PCR Assay B. Padmavathy • R. Vinoth Kumar • Amee Patel • S. Deepika Swarnam • T. Vaidehi • B. M. Jaffar Ali Received: 12 December 2011 / Accepted: 2 April 2012 / Published online: 22 April 2012 Ó Springer Science+Business Media, LLC 2012 Abstract Urinary tract infection (UTI) is among the most common bacterial infections and poses a significant health- care 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 per- formed 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 iso- lates 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. Introduction Urinary tract infection (UTI) is a prevalent infectious dis- ease with potentially severe complications especially in women of all age groups. The pathogens responsible for infection comprise 70–90 % of uropathogenic Escherichia coli (UPEC) and the remainder a variable contribution from Proteus, Klebsiella, Pseudomonas aeruginosa, Staphylococcus saprophyticus and Enterococcus. Numer- ous epidemiological studies have been performed on dif- ferent age groups to demonstrate the prevalence of UTI infections in humans [1–8]. The studies emphasize the demand for rapid clinical diagnosis of these pathogens. Various research groups have attempted to correlate uri- nary tract infection with the laboratory findings [9–12]. These well-performed evaluations suggest that quantitative urine cultures cannot be used to determine UTIs unless small numbers of organisms are identified and pyuria is measured. It should be noted that the detection sensitivity of these methods was at 10 5 cfu/ml. In addition, culture methods sometimes cannot reveal two or more organisms in the same culture medium if there is an overgrowth by a predominant species. Detection of UTI organism always remains an essential element in clinical diagnosis. Various research groups have evaluated the performance of rapid UTI detection kits [13–15]. These studies gave low specificity and high false- positive rate. The development of rapid screening tests and automated systems continues, but at present, microscopy and culture remain the most important techniques for lab- oratory diagnosis. Jolkkonen et al. [16] evaluated the detection of bacteria and leukocytes by a flow cytometer based urine analyzer, the UF-500i. They concluded that the analyzer could effectively reduce the workload for urine culture but the percentage of false-negative reported does B. Padmavathy Á R. Vinoth Kumar Á A. Patel Á S. Deepika Swarnam Á B. M. Jaffar Ali (&) AU-KBC Research Centre, M.I.T. Campus of Anna University, Chennai 600 044, India e-mail: jaffarali.bm@gmail.com T. Vaidehi Sundaram Medical Foundation and Dr. Rangarajan Memorial Hospital, Chennai 600 040, India Present Address: B. M. Jaffar Ali Centre for Green Energy Technology, Pondicherry University, Puducherry 605 014, India 123 Curr Microbiol (2012) 65:44–53 DOI 10.1007/s00284-012-0126-3