Real-Time Quantitative Broad-Range PCR Assay for Detection of the 16S rRNA Gene Followed by Sequencing for Species Identification

Abstract

Here we determined the analytical sensitivities of broad-range real-time PCR-based assays employing one of three different genomic DNA extraction protocols in combination with one of three different primer pairs targeting the 16S rRNA gene to detect a panel of 22 bacterial species. DNA extraction protocol III, using lysozyme, lysostaphin, and proteinase K, followed by PCR with the primer pair Bak11W/Bak2, giving amplicons of 796 bp in length, showed the best overall sensitivity, detecting DNA of 82% of the strains investigated at concentrations of <10 2 CFU in water per reaction. DNA extraction protocols I and II, using less enzyme treatment, combined with other primer pairs giving shorter amplicons of 466 bp and 342 or 346 bp, respectively, were slightly more sensitive for the detection of gram-negative but less sensitive for the detection of gram-positive bacteria. The obstacle of detecting background DNA in blood samples spiked with bacteria was circumvented by introducing a broad-range hybridization probe, and this preserved the minimal detection limits observed in samples devoid of blood. Finally, sequencing of the amplicons generated using the primer pair Bak11W/Bak2 allowed species identification of the detected bacterial DNA. Thus, broad-spectrum PCR targeting the 16S rRNA gene in the quantitative real-time format can achieve an analytical sensitivity of 1 to 10 CFU per reaction in water, avoid detection of background DNA with the introduction of a broad-range probe, and generate amplicons that allow species identification of the detected bacterial DNA by sequencing. These prerequisites are important for its application to blood-containing patient samples.

Gram Stain-Specific-Probe-Based Real-Time PCR for Diagnosis and Discrimination of Bacterial Neonatal Sepsis

“…1). These detection limits are among the lowest reported up to the present (24,34). In addition, multiple copies of the 16S rRNA gene were present in a single bacterial cell on the chromosomes of most bacteria in the GenBank database (NCBI).…”

“…The arbitrary definition of the clinically significant bacterial concentration was a C T value three C T values lower than the mean C T value from the negative template control. This definition was chosen to have a nearly 10-fold-higher concentration of detectable DNA in the positive samples than in the negative template control samples (34). In our assays, the negative template control showed C T values between 38 and 40.…”

Bodas de plata de la reacción en cadena de la polimerasa (PCR)

“…La concentración celular de ribosomas es proporcional a la síntesis de proteína total y de la actividad metabólica celular. Por lo cual es un buen marcador del contenido ribosomal, cuyos transcriptos tienen una estabilidad excepcional, ya que el 90% de todos ellos, durante la fase logarítmica, tienen una vida media de 5 minutos y estabilidad hasta por 3 horas (34,35,36).…”

Dual Priming Oligonucleotides for Broad-Range Amplification of the Bacterial 16S rRNA Gene Directly from Human Clinical Specimens

“…A positive specimen was defined as reaching the fluorescence threshold value (C T ) Ն3 cycles before the negative control (14,23). All specimens, both positives and negatives, were run until the amplification curve had reached the plateau level.…”

“…The finding of potentially relevant bacterial DNA upon sequencing in six of these PCR-negative samples reflects the reduced sensitivity for a universal PCR in samples containing low levels of pathogen DNA since a positive sample can only be reliably defined if it contains significantly more bacterial DNA than the negative control (i.e., the background/contaminant bacterial DNA from the reagents) (23). The small differences between pathogen DNA levels and background DNA levels in these specimens were demonstrated by background DNA being detectable as low secondary peaks in most of the chromatograms.…”