The authors have declared that no competing interests exist.
Conceived and designed the experiments: RJC DVZ PEW EPL PMG. Performed the experiments: RJC MM JP RQ YIK PMG. Analyzed the data: RJC PMG PEW EPL. Contributed reagents/materials/analysis tools: EPL PEW YIK. Wrote the paper: PMG RJC PEW EPL.
Within the paradigm of clinical infectious disease research,
In clinical microbiology laboratories, traditional culture based techniques remain the primary methodology used for identifying bacterial isolates. These methods are time-consuming, can involve multiple biochemical tests, and can be expensive, particularly for fastidious organisms. In recent years, the introduction of automated identification instruments has resulted in greater reliability, but discrepancies between the various platforms have been reported
Despite significant advances in molecular biology, molecular methods for species identification have not achieved widespread use, with the possible exception of the Biodefense community
Even prior to recent global calls to help combat the spread of carbapenem-resistant
We recently published a highly sensitive and specific real-time PCR assay for the detection of MRSA
Primer characteristics are presented in
Name |
Gene | Target species | Sequence (5′ to 3′) | Eff (%) |
Length |
U16SRT-F |
|
variable |
|
>96.4% | 180 |
U16SRT-R |
|
||||
secERT-F |
|
|
|||
secERT-R |
|
|
|
99.4 | 94 |
secERT-Probe |
|
|
|||
yccTRT-F |
|
|
|||
yccTRT-R |
|
|
98.1 | 59 | |
yccTRT-Probe |
|
||||
gltART-F |
|
|
|
97.1 | 68 |
gltART-R |
|
||||
ecfXRT-F |
|
|
|
93.8 | 81 |
ecfXRT-R |
|
Probe sequences were generated for the
Primer efficiency was calculated from the slope and intercept of the trendline produced following amplification of serial dilutions of genomic DNA from the ATCC strains of each species, as described previously
Amplicon size in base pairs.
To identify genes specific to
Four genes showed no significant match to non-
Starting with a multiple sequence alignment, a custom Perl script was used to identify positions within each gene that are invariant among all strains. With
The limited number of published genome sequences available for
All primer sets were tested for sensitivity, optimal annealing temperature and primer efficiency as previously described
Genome copies of DNA were calculated using the following formula:
Size of genome (in bp) × 650 Daltons/bp = molecular weight of Genome in g/mol.
# copies of genome in 1 ng of DNA = (1×10−9 g ÷ Mw of genome)×6.02×1023 molecules/mole (Avogadro’s number).
To get 108 copies in 2 µl = (108 ÷ # copies of genome in 1 ng)/2.
Serial dilutions of DNA from 108 to 102 copies were prepared and tested in duplicate with each primer set to calculate primer efficiency and sensitivity.
Primers were tested using two different instruments, the Roche Light Cycler 480 II (LC 480 II) with SYBR Green I Master Mix (Roche Applied Sciences, Indianapolis, IN), and the BioRad CFX96 with SsoAdvanced SYBR Green supermix (Bio-Rad Laboratories, Hercules, CA). Each primer was tested for specificity by two methods. First, the primers were tested against genomic DNA extracted from a panel of American Type Culture Collection strains (ATCC, Manassas, VA, USA) and clinical isolates representing fifty different bacterial species, including closely related members from the same genus (
The primers were incorporated into a 96-well plate assay to allow high throughput testing of multiple clinical isolates. All bacterial isolates were cultured overnight on Blood Agar plates at 37°C, and all samples were prepared in a BioSafety hood in a certified BSL2 laboratory. Single colonies from an overnight culture of each isolate were re-suspended in 200 µl of sterile, ultra-pure water and mixed by vortexing. 10 µl of the resulting suspension (or 10 µl taken directly from an overnight broth culture) was added to 20 µl of Lyse-and-Go reagent (Thermo Scientific, Waltham, MA) in 96-well plates, and run in a thermal cycler using the manufacturer’s protocol for the isolation of total genomic DNA. Isolates were held at 80°C for 15 minutes at the end of the program to maximize lysis, and 2 µl of the resulting lysate was used directly for real-time PCR. Leftover bacterial DNA in Lyse-and-Go reagent was stored at −20°C, and no reduction in real-time PCR amplification was evidenced after 9 months (data not shown). Appropriate positive (ATCC Type strains for each species), negative (two ATCC type strains from species other than the target organism), and no template controls (ultra-pure water) were incorporated onto every plate. Cycling parameters were 95°C for 5 minutes, followed by 40 cycles of 95°C for 10 seconds and 56°C for 10 seconds. A melting curve analysis was included at the end of every program to assist in data analysis. Quantification cycle (Cq; CFX96) and crossing threshold (Ct; LC 480 II) values were calculated automatically using instrument software.
(
Analysis of the alignment of 962,279 bacterial
Primers were tested against serial dilutions of genomic DNA from
The
Five sets of primers were tested for sensitivity and specificity for
Four sets of primers were tested for sensitivity and specificity for
The final two primers, targeting the
Despite 38 sequenced genomes of
The paucity of completed genomic sequences for both
BLAST analysis suggested a potential cross-reaction between the
Both sets of
Species-specific primers targeting the
We describe a set of real-time PCR primers, designed to have the same optimal annealing temperature, and displaying high specificity for four clinically important pathogens. The primers are well suited for high-throughput testing of isolates, with results available in less than 90 minutes from bacterial colonies or overnight broth cultures. We also demonstrate the power of bioinformatics in designing optimal primer sequences, and provide a novel
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