PCR-based Method Rapidly Detects Pathogens in the Environment

A newly developed PCR-based technique detects soil, air, and waterborne pathogens within 24 hours, which is three to four days faster than currently used culture-based methods.

The new method, which was developed by investigators at Ben-Gurion University of the Negev (Be'er Sheva, Israel) and the Massachusetts Institute of Technology (Cambridge, MA, USA), combines state-of-the-art quantitative PCR with the classical probable number (MPN) estimation method.

The MPN method is a well-established and fully documented method of estimating the number of viable microorganisms in product in which the microorganisms are randomly distributed. Its principal uses are in the food and water industries where it is used with liquid, powdered, and semi-solid products or raw materials. The MPN method consists of taking replicate samples of product (by volume or weight) which contain, on average, the same number of viable microorganisms in each of them (hence the requirement for randomness of distribution) and scoring each sample individually for the presence of viable microorganisms by means of transferring to liquid growth media and incubation. From the frequency of the occurrence of positive tests within a set of replicates, an estimate is made of the number of viable microorganisms present in the sample or the bulk product from which the sample has been taken. The estimate and its confidence limits are then derived from published MPN tables.

In the new technique, transfer to growth media and time-consuming incubation were replaced by quantitative PCR, which allowed rapid screening for presence of the amplicon (the bacterial target).

In the current study, a variety of environmental samples, including aerosols, soil of various types (sand, sand/clay mix, and clay), wastewater, and vegetable surface (modeled by a tomato), were concomitantly spiked with Salmonella enterica and/or Pseudomonas aeruginosa to determine recovery rates and limits of detection. The various matrices were first enriched with a general pre-enrichment broth in a dilution series and then enumerated by most probable number (MPN) estimation using quantitative PCR for rapid screening. Soil and aerosols were then tested in non-spiked environmental samples, as these matrices are prone to large experimental variation.

Results published in the August 18, 2015, online edition of the journal Water, Air & Soil Pollution revealed that the limit of detection in the various soil types was one to three colony-forming units (CFU). In other samples the limits were: one CFU per gram on vegetable surfaces, five CFU per tomato, five CFU per liter in treated wastewater, and more than 300 CFU per milliliter in aerosols.

The method accurately identified S. enterica in non-spiked environmental soil samples within a day, while traditional methods took four to five days and required sorting through biochemically and morphologically similar species. Likewise, the method successfully identified P. aeruginosa in non-spiked aerosols generated by a domestic wastewater treatment system.

First author Dr. Ezra Orlofsky, a researcher in the institute for water research at Ben- Gurion University of the Negev, said, "Rapid and reliable pathogen detection in field samples is critical for public health, security and environmental monitoring. Current methods used in food, water, or clinical applications rely on labor and time-intensive culturing techniques while activities such as dairy farming, wastewater, and runoff treatment necessitates real-time monitoring of pathogens in environment samples. This is the first study to comprehensively assess pathogen concentrations in such a broad variety of environmental sample types while achieving multiple pathogen detection with complete parallel testing by standard (or traditional) methods."

Related Links:
Ben-Gurion University of the Negev
Massachusetts Institute of Technology