The microbial screening workflow consists of first extracting the nucleic acids from the sample of interest and loading these extracts onto a 96-well plate. Upon PCR amplification, the plate is then loaded on the PLEX-ID system. Each sample is run through an automated clean up process and injected into the mass spectrometer portion of the instrument to determine the molecular weight for each sample well. The software algorithm then converts the primer pairs from each well to a base composition and compares all the results obtained for each sample to the database to identify the organisms present. Up to eight or more wells are used per sample with the actual number of wells used dependent upon the breadth of coverage required. The overall microbial screening workflow typically takes between six and eight hours from sampling to result, depending upon the amount of extraction and amplification required. This includes an instrument run time of approximately 70 minutes per plate.
The steps from sample extraction to result include:
- Extraction of the nucleic acids from all types of organisms present
- Applying each sample to specific wells of the 96-well plate according to the plate layout
- Performing traditional PCR to amplify the sequence regions specific to the organisms of interest
- Loading the 96-well plate on the PLEX-ID to desalt the contens of each sample well and determine the mass of each amplified product (amplicon) present
- Automatically calculating the base composition of number of A, C, G, and aT bases from the mass of each amplicon present
- Automatically performing a database search by for all amplicons against a library greater than 750,000 base composition entries to determine and report the organisms present
