Computational protocol: Multiplex polymerase chain reaction of genetic markers for detection of potentially pathogenic environmental Legionella pneumophila isolates

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Protocol publication

[…] This study was conducted in the department of Microbiology, All India Institute of Medical Sciences, New Delhi, India during 2012-2015. The study was approved by the Institutional Bio-Safety Committee (water and swab samples - 200 each approved for collection). No human subjects and/or animals were involved in this study.Culture and DNA extraction: The standard strains ATCC 33152 L. pneumophila strain Philadelphia-1 and ATCC 33153 L. pneumophila strain Knoxville were grown on buffered charcoal-yeast extract agar (BD-BBL, France) supplemented with 4 per cent L-cysteine-HCl (HiMedia laboratories, Mumbai) in a candle jar incubated at 37°C for three days. Gram stain was done and blood agar was used as a negative control plate. DNA was extracted from the colonies by the boiling method. Briefly, colonies were boiled in sterile distilled water for 10 min and centrifuged at 10,000 ×g to get the supernatant having the DNA.PCR analysis and restriction enzyme analysis (REA): PCR detection of the 16S rRNA gene and REA were used for identification and confirmation of L. pneumophila standard strains and environmental isolates. The restriction enzyme TaaI (HpyCH4III endonuclease, Thermo-Scientific Ltd., USA) was used for the differentiation of L. pneumophila and non-pneumophila Legionella species. Briefly, 15 μl of PCR product was digested with ×10 buffer Tango, 1U TaaI enzyme and adjusted with sterile water to final volume of 60 μl. The reaction was carried out in a water bath at 65°C for one hour and 20 μl of digested products were checked using 1.5 per cent agarose gel electrophoresis.Multiplex PCR, sensitivity and specificity: Standardization of multiplex PCR for detection of five markers annotated to L. pneumophila plasmid pLPP (11A2), lipopolysaccharide synthesis (19H4), CMP-N-acetylneuraminic acid synthetase (10B12), conjugative coupling factor (24B1) and hypothetical protein (8D6) was done using the available gene sequences (Acc.nos. HM584933-HM584937). Primers () were selected and designed using NCBI Primer BLAST programme, and MPprimer design software was used for multiplex PCR primer design. The primers were synthesized commercially (Sigma, USA) and the DNA from both the standard strains were used for the multiplex PCR. Briefly, each reaction mix contained 5 μl of DNA, 5 μl of ×10 PCR buffer (Bangalore Genei, India), 1.5 μl of 10 mmol dNTP mix (Thermo Scientific), 0.5 μl of 10 pmol/μl each of five sets of primer, 0.5 μl of 3 U/μl Taq polymerase (Bangalore Genei) and sterile water adjusted to a final volume of 50 μl. The reaction was carried out in a thermal cycler (Applied Biosystems, USA) with the thermal programme having initial activation of 94°C for five minutes, 30 cycles of 94°C, 56°C, 72°C for 30 sec each and final extension of 72°C for 10 min. The primer pairs were checked individually for their sensitivity using various dilutions of standard strains’ DNA and specificity with DNA isolated from various bacteria. The individual PCR products were purified using QIAquick Gel extraction kit (Qiagen, Germany) and sequenced commercially to confirm their identity.Hospital environmental sample collection and processing: A total of 145 water samples (both potable drinking water and non-potable water used for bathing, washing, gardening, cooling tower water) and 200 swab samples (pre-water collection) were collected and processed from July 2012 to May 2014. The samples were collected from distal outlets and from AC cooling towers (basin beneath the tower). Temperatures of the samples during the collection were also noted using a mercury thermometer (Zeal, England). The samples were treated with 0.1 N sodium thiosulphate and filtered with 0.22 μm mixed cellulose ester filters (Millipore, Germany). The filtrate was subjected to acid and thermal treatment and processed further for bacteriologic examination ().Multiplex PCR of environmental isolates: The multiplex PCR for five markers was applied as mentioned above to randomly chosen 45 hospital environmental isolates of L. pneumophila and one non-pneumophila Legionella species. Electrophoresis of the products was done in 1.5 per cent agarose gel stained with ethidium bromide, visualized under UV light and gel documentation system (Gel Doc EZ imager, Bio-Rad, USA). The presence or absence of the band was compared with the bands of standard strain ATCC 33153. […]

Pipeline specifications

Software tools Primer-BLAST, MPprimer
Application qPCR
Organisms Legionella pneumophila, Saccharomyces cerevisiae
Chemicals Cysteine