PCR inhibitors are one of the most common causes of false negative results in pathogen detection. This is especially true with assays that are sold commercially such as Direct PCR . For the PCR amplification and detection to work, PCR inhibitors must be removed and/or neutralized without degrading the target DNA. Our extraction and purification protocol does both.
Sample Type: Urine
There are several PCR inhibitors in urine such as urea, beta-human chorionic gonadotropin, and crystals. We developed a 20-minute extraction and purification protocol that removes PCR inhibitors from urine. We spiked nuclease free water sample (no inhibitors) and urine sample with an equal amount of target DNA sequence. We ran our extraction, purification and probe-based PCR protocol on the spiked-samples. The result in the Table 1 below shows that our method removes inhibitors from urine with cq values that are identical to inhibitor-free nuclease free water.
Statistical Analysis: A paired t-test was performed. p-value was greater than 0.05 at 0.99. Null hypothesis is accepted.
Conclusion: Our proprietary extraction protocol removes PCR inhibitors from Urine.
Sample Type: Plasma and Vaginal Swab
There are several PCR inhibitors in plasma such as immunoglobulin G, lactoferrin, bilirubin, bile salts, EDTA. Similarly, vaginal swab has several inhibitors. We spiked nuclease free water sample (no inhibitors) , 100% plasma and vaginal swab sample with an equal amount of target DNA sequence. We performed our PCR protocol on these samples. Results are shown in Table 2.
Table 2.
Statistical Analysis: A one-way ANOVA was performed. F statistic was 1.38 which is less than F critical value of 3.55. p-value was greater than 0.05 at 0.276. Null hypothesis i.e there is no difference in the means is accepted.
Conclusion: Our proprietary extraction protocol removes PCR inhibitors from plasma as well as vaginal Swab.