MOLECULAR DETECTION AND IDENTIFICATION OF ASPERGILLUS NIGER CONTAMINATION IN COSMETIC/PHARMACEUTICAL RAW MATERIALS AND FINISHED PRODUCTS

Abstract

Abstract A PCR-based assay using a one step sample preparation was developed and validated against standard methods for the rapid detection of Aspergillus niger contamination in cosmetic/pharmaceutical raw materials and finished products. Artificially contaminated samples were added to Sabouraud Dextrose Broth (SDB) with 10% Tween 20 and 3% Soy Lecithin. Samples were then agitated at 200 revolutions per minute for 24 h at 35C. Prior to the PCR assay, sample preparation consisted of adding 10 μL of the 24 h enrichment broth sample mixture to a lysis buffer containing Tris-EDTA and 0.4% SDS. DNA extraction was accomplished by boiling for 1 h to release the mycelial DNA. After DNA extraction, a 50 μL aliquot of the lysate was then added to a PCR tube containing the following ingredients: 4 Ready-To-Go PCR beads, 2 μL of two fungal primers, and 48 μL of sterile water. The DNA primers encoded for specific sequences of the Aspergillus spp. 18S rRNA gene. A 363 bp DNA fragment was detected in all of the artificially contaminated samples. Standard methods require 6–8 days to complete the isolation and identification of mold contamination. However, the PCR-based assay was completed within 27–30 h. Rapid detection of mold contamination in cosmetic/pharmaceutical raw materials and finished products can be used to optimize the quality evaluation of a sample to allow the release in 27–30 h.