Loop-Mediated Isothermal Amplification (LAMP) Assay for Detecting Burkholderia cepacia Complex in Non-Sterile Pharmaceutical Products

Abstract

Simple and rapid detection of Burkholderia cepacia complex (BCC) bacteria, a common cause of pharmaceutical product recalls, is essential for consumer safety. In this study, we developed and evaluated a ribB-based colorimetric loop-mediated isothermal amplification (LAMP) assay for the detection of BCC in (i) nuclease-free water after 361 days, (ii) 10 μg/mL chlorhexidine gluconate (CHX) solutions, and (iii) 50 μg/mL benzalkonium chloride (BZK) solutions after 184 days. The RibB 5 primer specifically detected 20 strains of BCC but not 36 non-BCC strains. The limit of detection of the LAMP assay was 1 pg/μL for Burkholderia cenocepacia strain J2315. Comparison of LAMP with a qPCR assay using 1440 test sets showed higher sensitivity: 60.6% in nuclease-free water and 42.4% in CHX solution with LAMP vs. 51.3% and 31.1%, respectively, with qPCR. These results demonstrate the potential of the ribB-based LAMP assay for the rapid and sensitive detection of BCC in pharmaceutical manufacturing.

A Propidium Monoazide (PMAxx)-Droplet Digital PCR (ddPCR) for the Detection of Viable Burkholderia cepacia Complex in Nuclease-Free Water and Antiseptics

“…Two primers set RibB5 and RibB67 were chosen because of their specificity and sensitivity in BCC detection as reported previously [ 30 ]. To evaluate the efficacy of the two primer sets, 20 BCC strains and 36 other bacterial strains (i.e., 18 non-BCC, and 18 non- Burkholderia ) were initially examined ( Supplementary Table S1 ).…”

“…In fact, BCC microorganisms have a recA gene sequence comparability of 94 to 95% between species and 98 to 99% between sequences of the same species, making it a viable candidate for species identification [ 2 , 10 ]. The ribB -based BCC-specific loop-mediated isothermal amplification assay (LAMP) primers (RibB5, RibB16, and RibB67) were previously developed [ 30 ]. Both BLAST analysis and full-scale lab trials were used to confirm their specificity and sensitivity.…”

“…The 20 BCC, 18 non-BCC species and 18 non- Burkholderia bacterial strains were used in this research as described previously ( Table S1 in the Supplemental Materials ) [ 9 , 30 ]. The total genomic DNA was extracted from cultures of the aforementioned test strains using the DNeasy UltraClean Microbial Kit (Qiagen, Valencia, CA, USA), as described previously [ 30 ].…”

“…The 20 BCC, 18 non-BCC species and 18 non- Burkholderia bacterial strains were used in this research as described previously ( Table S1 in the Supplemental Materials ) [ 9 , 30 ]. The total genomic DNA was extracted from cultures of the aforementioned test strains using the DNeasy UltraClean Microbial Kit (Qiagen, Valencia, CA, USA), as described previously [ 30 ]. The primer sets used for ddPCR in this study are RibB5-F/RibB5-R and RibB67-F/RibB67-R ( Table 1 ), which have proven effective for amplifying the ribB genes from all bacterial species tested [ 30 ].…”

“…Genomic DNA was extracted using 100 µL sample from the dilutions by boiling at 100 °C for 10 min in a water bath, and 4 µL of DNA were used for the ddPCR assay. Based on the fluorescence amplitude of PMAxx-ddPCR, positive results were recorded (0 = not detected, 1 = detected), and the estimated sensitivity was calculated [ 30 ].…”