Marijuana Business Magazine July 2019

Marijuana Business Magazine | July 2019 90 10 Plate Samples for qPCR Analysis This part of the process requires meticulous work and involves a quantitative polymerase chain reaction (qPCR) method. Rm3 Labs’ qPCR method uses a specific DNA sequence present in all species of shiga toxin-producing E. coli and salmonella that is attached to a fluorescent probe to detect if these bacteria are present in samples The target sequence is not shared by other bacteria or organisms. After the DNA samples are extracted, an analyst pipettes the sample into a 96-well plate. Inside each well is the qPCR chemistry that allows for the detection of a target DNA sequence. qPCR reaction chemistries can be purchased in kits from manufacturers, but Rm3 Labs has developed its own. That cuts costs, which reduces the prices for some tests. Remember those quality-control samples mentioned earlier? The DNA from those samples is also extracted and plated for qPCR analysis alongside client samples. If the “positive” quality-control samples produce a positive result during qPCR, this tells the lab that the correct procedures were used to extract DNA from all samples in the batch and assures the lab that client samples producing a negative result are truly negative, ruling out any false negatives. Other quality controls are added to the qPCR plate to ensure the qPCR reaction chemistries were prepared appropriately. 11 Load the qPCR Machine The 96-well plate with samples is loaded into the qPCR machine. The plate is repeatedly heated and cooled over 45 minutes to amplify, or increase, the DNA in the sample. Heating the plate breaks apart the two strands of any DNA present in the samples, and cooling allows for newly assembled DNA strands to anneal—in other words, be brought back together. During this process, if the target DNA sequence of the bacteria of interest is present, the probe attached to the target DNA sequence will cause a fluorescent signal on the qPCR machine. As the DNA of interest is amplified, the probe’s fluorescence is also increased, and this is shown in an S-shaped, curved line. The sooner the curved line pops up on the screen, the more concentrated the DNA of the bacteria of interest is within the sample. In the tens of thousands of samples that Rm3 Labs has tested, the laboratory has detected salmonella in only one sample. The curved lines on the screen show the results for positive quality control samples. All other lines are flat, which is what an operator hopes to see in client samples because it indicates no bacteria of interest is present. A Day in the Life