STUDY ON RT-QPCR TECHNIQUE FOR QUANTIFICATION OF BILE-TOLERANT GRAM-NEGATIVE BACTERIA IN PHARMACEUTICALS

Abstract

Backgrounds: According to the Vietnamese Pharmacopoeia V, the bile-tolerant Gram-negative bacteria index is a mandatory requirement for testing the bacterial limits of pharmaceuticals originated from animal, plant, mineral and for inhaled medication. RT-qPCR technique features rapid determination of the number of bile-tolerant Gram-negative bacteria based on specific genes with high sensitivity and accuracy.

Objectives: Applying the RT-qPCR technique to quantify the total number of bile-tolerant Gram-negative bacteria.

Methods: Primers and probes were designed in silico and tested for feasibility in vitro. The method of quantifying the total number of bile-tolerant Gram-negative bacteria using the RT-qPCR technique was evaluated for validation criteria, including specificity, linearity, accuracy, the limit of quantification (LOQ), and the limit of detection (LOD).

Results: The RT-qPCR method for quantifying the total number of bile-tolerant Gram-negative bacteria using the primer and probe combinations, namely rplP_F1/R1/P1 and oprI_F1/R1/P1 with primer and probe concentrations of 400 and 200 nM, respectively, met the requirements for specificity, linearity, and accuracy (RSD < 25%), with LOQ of 4.102 cells/ml and LOD at 121 cells/ml.

Conclusions: The RT-qPCR technique can be applied to test the total number of bile-tolerant Gram-negative bacteria required to determine the limit of contamination in pharmaceutical samples.

Keywords: bile-tolerant Gram-negative bacteria; Enterobacteriaceae; Pseudomonas; bacterial limits