Two Novel Real-Time Reverse Transcriptase PCR Assays for Rapid Detection of Bacterial Contamination in Platelet Concentrates

Abstract

The incidence of platelet bacterial contamination is approximately 1 per 2,000 units and has been acknowledged as the most frequent infectious risk from transfusion. In preliminary studies, the sterility of platelet concentrates (PCs) was tested with an automated bacterial blood culturing system and molecular genetic assays. Two real-time reverse transcriptase PCR (RT-PCR) assays performed in a LightCycler instrument were developed and compared regarding specificity and sensitivity by the use of different templates to detect the majority of the clinically important bacterial species in platelets. Primers and probes specific for the conserved regions of the eubacterial 23S rRNA gene or the groEL gene (encoding the 60-kDa heat shock protein Hsp60) were designed. During the development of the 23S rRNA RT-PCR, problems caused by the contamination of reagents with bacterial DNA were noted. Treatment with 8-methoxypsoralen and UV irradiation reduced the level of contaminating DNA. The sensitivity of the assays was greatly influenced by the enzyme system which was used. With rTth DNA polymerase in a one-enzyme system, we detected 500 CFU of Escherichia coli or Staphylococcus epidermidis/ml. With a two-enzyme system consisting of Moloney murine leukemia virus RT and Taq DNA polymerase, we detected 16 CFU/ml. With groEL mRNA as the target of RT-PCR under optimized conditions, we detected 125 CFU of E. coli/ml, and no problems with false-positive results caused by reagent contamination or a cross-reaction with human nucleic acids were found. Furthermore, the use of mRNA as an indicator of viability was demonstrated. Here we report the application of novel real-time RT-PCR assays for the detection of bacterial contamination of PCs that are appropriate for transfusion services.

High-Volume Extraction of Nucleic Acids by Magnetic Bead Technology for Ultrasensitive Detection of Bacteria in Blood Components

“…We optimized our method for detecting bacterial contamination in PCs with abundant 23S rRNA to obtain the highest achievable assay sensitivity, specificity, and rapidity (14 ). The simultaneous extraction of total nucleic acids should impart increased sensitivity.…”

“…The probe and primers for amplifying a 122-base pair fragment of bacterial 23S rRNA were based on a previously published alignment of 23S rRNA sequences (14 ). As an IC, we coextracted and coamplified human Table 2 in the online Data Supplement), 200 nmol/L 23S fluorescent probe, 200 nmol/L IC fluorescent probe, and 2.5 U rTth polymerase.…”

“…We tested 2 other probe systems in the evaluation study-the conventional TaqMan probe (14 ) and a minor groove binder (MGB) group (see LNA sequence) probe (Applied Biosystems)-to detect the bacterial nucleic acids at the same high temperature (60°C). In contrast to conventional TaqMan probes, MGB probes contain an MGB with a nonfluorescent quencher.…”

“…Our focus was on minimizing sampling error and increasing the sensitivity of our broad-range 23S rRNA real-time reverse transcription (RT)-PCR assay (14 ). We enhanced the sensitivity of the 23S rRNA RT-PCR assay for detecting bacterial contamination of PCs by modifying sample volume, nucleic acid input, probe technology, and the nucleic acid extraction method.…”

“…Reflecting these concerns, the American Association of Blood Banks implemented a directive stating that blood banks and transfusion services shall have methods to limit and detect bacterial contamination in all platelet components after March 2004 (10,11 ). The different methods for bacterial screening can be categorized into culture and rapiddetection methods (12)(13)(14)(15)(16)(17). Culture methods, which require a long time to demonstrate the presence of bacteria, have remained the preferred way for platelet bacteria screening, although bacteria in PCs have been detected with other approaches (18 ).…”