NGHIÊN CỨU KỸ THUẬT REAL-TIME PCR ĐỂ KIỂM TRA CHỈ TIÊU TỔNG SỐ VI KHUẨN HIẾU KHÍ TRONG GIỚI HẠN NHIỄM KHUẨN CỦA DƯỢC PHẨM

Abstract

Background: Microbiological testing is a mandatory requirement for pharmaceuticals being put into circulation on the market. Real-time PCR is a technique that enables rapid detection of microorganisms present in a sample with high sensitivity and precision. Objectives: This research aims to study real-time PCR techniques for examining aerobic mesophilic bacteria criteria of bacterial limits. Methods: The RNA extraction method using SDS-TRIzol was investigated on Gram-positive and Gram-negative bacterial strains. For each criterion on determining the bacterial limits by real-time PCR, the primers were checked in silico and in vitro, selected at the appropriate concentration. The method of quantifying microorganisms using real-time PCR was validated for specificity, linearity, precision, accuracy, the limit of quantification (LOQ), and the limit of detection (LOD). Results: The RNA extraction method using SDS-TRIzol gives the best RNA extraction efficiency with a heat treatment time to break down bacterial cell walls of 5 minutes and does not require treatment with lysozyme. Method to quantify the total number of aerobic mesophilic bacteria using RT-qPCR technique with the pair of primers Bac_890F/ Bac_1034R at a concentration of 50 nM had LOQ and LOD values of 8 CFU/ml and 2 CFU/ml, respectively. The examination method met the validation requirements of specificity, linearity, precision, and accuracy (RSD < 25%). Conclusions: The real-time PCR technique could be applied to examine aerobic mesophilic bacteria criteria of bacterial limits in pharmaceuticals.