The 16s ribosomal ribonucleic acid microorganisms’ detection in mesenteric lymph nodes by a polymerase chain reaction in view of colorectal cancer

Abstract

Objective: This study proposes a method to detect 16s rRNA microorganisms in mesenteric lymph nodes (MLN) using a polymerase chain reaction (PCR) in patients with colorectal cancer (CRC). Material and methods: To quantify the presence of microorganisms in MLN, it is proposed to determine the dependence of the accumulated amplification products on the number of colony-forming units of bacteria (CFU/ml). The pure culture of Escherichia coli, GFP 6 serotype of biotype 1 (ATCC® 25922GFP™) with the CFU values from 102 to 108 (group 1) as well as the mixtures of E.coli with CFU/ml from 102 to 108 with the MLN tissues (group 2) were calibrated. The third group consisted of the MLN patients (60 people) with CRC without bowel obstruction. The 16s rRNA bacteria in MLN was detected by using real-time PCR by the BIO-RAD CFX96 amplifier. Results: To assess the dependence of the bacteria’s CFU/ml logarithm on the value of the threshold cycle amplification, a model was developed in the form of an equation. The amplification curves, threshold cycle values, and PCR efficiency differ from the first two groups. This can be due to the presence of DNA amplification-inhibiting compounds as well as the non-specific binding of MLN primers to DNA. Therefore, a mathematical model of the second group (suspension of E.Coli and MLN) was used to study the translocation of microorganisms in MLN. According to the developed mathematical model, depending on the values of the threshold amplification cycles, the positive PCR result in the study group (patients with CRC) was detected in 15 patients (25%). At the same time, the level of CFU/ml with bacterial translocation in MLN does not exceed 104. Conclusion: The developed method allows to determine the microbial DNA in MLN quantitatively in a wide range of its concentrations (102 to 108 CFU).