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The Technology of Microgen DX: qPCR for rapid Screening, Gene Resistance & Next-Gen Sequencing

qPCR Panel Rapid Screening

The qPCR Panel is a quantitative real-time PCR test for bacteria and fungi. The Panel also includes a qualitative real-time PCR test for resistance factors, vancomycin, methicillin, beta lactam, carbapenem, macrolide, aminoglycoside, tetracycline, and quinolone.

The Panel utilizes unique genes present in each organism to identify how much of that organism is present in each patient sample. This concentration is achieved in a multistep process. First, each gene was specifically chosen for its ability to distinguish the bacterial or fungal targets from closely related species and its abundance in the cells themselves (1 gene per bacterial or fungal cell). Each target was then measured to get an initial concentration (cfu/mg or mL and DNA concentration ng/uL after DNA extraction is performed). The initial concentration of cfu/mg or mL is also used to evaluate and calculate the extraction efficiency of each bacteria and fungi. The extraction efficiency percentage is taken into account when calculating the target analyte’s concentration in order to negate the slight loss of cells during the extraction process. Next, each initial target bacterial or fungi DNA, whose concentration was measured to obtain an initial concentration (ng/uL), was diluted to obtain a six to eight-fold serial dilution series and run on the quantitative PCR (qPCR) panel assay on the Roche LightCycler 480 II instrument. The dilution series was designed to span the entire analytic range (very high to very low concentrations). The dilution series concentration and cycle threshold generated a standard curve for each target. Based on this standard curve, the concentration for each target can be calculated.

The wound, ENT, UTI, and nail panel will detect 17 microbial organisms and resistance genes that may be present in patient specimens. These include the following analytes numbered 1-17, 4-20, 2-4 6-17 21-22, and 2 4-17 23 respectively.

The above analytes, except for methicillin, vancomycin, beta lactam, carbapenem, macrolide, tetracycline, quinolone, and 16S, are performed via quantitative assay, which will report out the organisms as copy per mL or mg. The resistance factors are performed via qualitative assay and reported as either present or absent. The 16S universal bacteria is a semi-quantitative assay, which reports the total bacteria load as low, medium, and high. These ranges for general bacteria copy number per mL or mg are <1E5 or <105, 1E5-1E7 or 105-107, and >1E7 or >107 for low, medium, and high respectively. The ranges are also used for swab samples since swabs cannot be weighed accurately.

The sensitivity and specificity of the assay are 100% on E. faecalis, E. faecium, K. pneumoniae, P. aeruginosa, S. aureus, S. pyogenes, vanA, C. albicans, M. catarrhalis, E. coli, P. mirabilis, T. rubrum, and S. pneumoniae. The sensitivity and specificity for S. agalactiae are 100% and 99.4%. The sensitivity and specificity for mecA are 100% and 95.2% and for 16S are 100% and 97.1%. The sensitivity and specificity for H. influenzae are 95.0% and 100%.

The accuracy of the above assays is 100% on all except, mecA, 16S, and H. influenzae. The accuracy is 98.9%, 99.6%, and 99.6% for mecA, 16S, and H. influenzae respectively.

MicroGen DX Laboratory will notify all clients of any changes to methods or procedures that may change the interpretation of data.

Gene Resistance Detection Information in our PCR Rapid Screen Test

Quinolone Level 1:

Level 1 test for Quinolone resistance is a real-time qualitative PCR assay utilizing the Roche LightCycler 480. This taqman assay uses dye labeled hydrolysis probes to detect the presence of a unique sequence in the qnr and gyrA gene specific to quinolone resistance. Quinolone antibiotics inhibit bacterial replication by stabilizing the complex between DNA and DNA gyrase or topoisomerase and blocking the polymerase from binding during replication. Quinolone resistance occurs when the DNA gyrase or topoisomerase is protected against the cleaving caused by Quinolone antibiotics. Qnr is a common quinolone resistance gene that blocks the quinolone antibiotics from inhibiting the DNA gyrase activity. A mutation in the DNA gyrase subunit A, gyrA, causes the quinolone antibiotics to become ineffective at stopping the DNA gyrase replication. The accuracy, sensitivity, and specificity of the qPCR assay are 100%. The limit of detection is 0.001ng/uL.

Methicillin Level 1:

Level 1 test for methicillin resistance is a real-time qualitative PCR assay utilizing the Roche LightCycler 480.  This taqman assay uses dye labeled hydrolysis probes to detect the presence of a unique sequence in the mecA genes specific to methicillin resistance. The mecA gene is the most common gene used to detect methicillin resistance among Staphylococcus species.  The accuracy, sensitivity, and specificity of the qPCR assay are 98.9%, 100%, and 95.2%.  The limit of detection is 0.0147ng/uL.

Vancomycin Level 1:

Level 1 test for vancomycin resistance is a real-time qualitative PCR assay utilizing the Roche LightCycler 480.  This taqman assay uses dye labeled hydrolysis probes to detect the presence of a unique sequence in the vanA genes specific to vancomycin resistance. The vanA gene is the most common mutated gene used to detect vancomycin resistance.  The accuracy, sensitivity, and specificity of the qPCR assay are 100%.  The limit of detection is 0.0005ng/uL.

Beta-lactam Level 1:

Level 1 test for beta-lactam resistance is a real-time qualitative PCR assay utilizing the Roche LightCycler 480.  This taqman assay uses dye labeled hydrolysis probes to detect the presence of a unique sequence in the TEM and SHV genes specific to beta-lactam resistance. TEM-1 is the most common gene used to detect beta-lactamase resistance due to the majority of beta lactamase resistant bacteria targets the TEM group to cause resistance.  The second most common group targeted is the SHV group.  The accuracy, sensitivity, and specificity of the qPCR assay are 100%.  The limit of detection is 0.0034ng/uL.

Carbapenem Level 1:

Level 1 test for Carbapenem resistance is a real-time qualitative PCR assay utilizing the Roche LightCycler 480.  This taqman assay uses dye labeled hydrolysis probes to detect the presence of a unique sequence in the blaKPC, blaNDM, and blaOXA48 genes specific to carbapenem resistance.  Carbapenem resistance can be divided into several groups including NDM, KPC, OXA48, IMP, and VIM; however, the most common groups found in the USA are NDM, KPC and OXA48.  NDM resistance is typically found in Esherichia coli and Klebsiella pneumoniae, while KPC is common to Klebsiella species and Pseudomonas aeruginosa.  OXA 48 resistance is found primarily in Acinetobacter species.  The accuracy, sensitivity, and specificity of the qPCR assay are 100%.  The limit of detection 3.36ng/uL.

Macrolide Level 1:

Level 1 test for macrolide resistance is a real-time qualitative PCR assay utilizing the Roche LightCycler 480.  This taqman assay uses dye labeled hydrolysis probes to detect the presence of a unique sequence in the ermB gene specific to macrolide resistance and is the most common route to macrolide resistance.  The accuracy, sensitivity, and specificity of the qPCR assay are 100%.  The limit of detection is 0.014ng/uL.

Aminoglycoside Level 1:

Level 1 test for Aminoglycoside resistance is a real-time qualitative PCR assay utilizing the Roche LightCycler 480.  This taqman assay uses dye labeled hydrolysis probes to detect the presence of a unique sequence in the aacC6-aph3 and ant-la-aph2 genes specific to aminoglycoside resistance.  The most common enzymes causing aminoglycoside resistance target the amino groups using N-acetyltransferases (AAC), O-nucleotidyltransferases (ANT), and O-phosphotransferases (APH).  The accuracy, sensitivity, and specificity of the qPCR assay are 100%.  The limit of detection is 0.0034ng/uL.

Tetracycline Level 1:

Level 1 test for Tetracycline resistance is a real-time qualitative PCR assay utilizing the Roche LightCycler 480.  This taqman assay uses dye labeled hydrolysis probes to detect the presence of a unique sequence in the tetM and tetB gene specific to tetracycline resistance.  Tetracycline resistance occurs when proteins stop binding the tetracycline to the bacterial ribosome.  There are 14 proteins that can block the binding site, but four cause the majority of blockage.  The most common protein found is tetM (at 80%).  The proteins tetA and tetB were identified first, but their mechanism is utilized in many of the same bacteria (10%).  The protein tetO is the newest discovered at less than 5% and many identified outside the USA.  The other 10 proteins make up the remaining 5%.  The accuracy, sensitivity, and specificity of the qPCR assay are 100%.  The limit of detection is 0.014ng/uL.

Next-Gen Sequencing Comprehensive DNA Test

The Comprehensive DNA test will detect virtually all microbial organisms and fungal pathogens that may be present in patient specimens. Microbial DNA in each sample will be sequenced using the Ion Torrent PGM sequencer in order to establish what type of bacterial and fungal species are present. Forward and reverse primers are used to detect and amplify the target sequence in each sample. The samples are differentiated from each other when run on the PGM sequencer by a "tag," a unique identifying sequence attached to the forward primer implemented when the targeted sequence is amplified using polymerase chain reaction or PCR.

The Ion Torrent PGM sequencer uses DNA polymerase to incorporate phosphorylated dNTPs into the DNA strand. When each dNTP is incorporated, a Hydrogen Ion or Hydron is released. The Hydron causes a change in pH proportional to the number of nucleotides incorporated in the DNA strand and is captured by sequencer’s sensing layer inside the chip.

The sequencing reads are analyzed for quality and length. Any reads that fall below the quality score or appropriate length are discarded due to the poor quality. Only the high quality reads will be interpreted and reported to physicians. Data for each detected bacterial or fungal species IS REPORTED as Relative Percent of total SPECIMEN reads.

This assay is capable of detecting the organisms listed here from as little as 100ng of patient specimen. Every species can be accurately detected with as little as 10ng/µL of extracted DNA with at least 1,000 copies of each species. Each organism is reported as a relative abundance in the sample.

174 ATCC species were run on bacteria and fungi sequencing assays to confirm accuracy, sensitivity, and specificity. All ATCC species matched their intended targets. 90 patient samples were run on both assays twice on two different sequencers to test the precision of the Ion Torrent PGMs. All patient samples were compared for read count and species identification using an analysis of variance. In addition to these measures of quality, both assays’ sensitivity/limit of detection is verified every 6 months using a dilutions series of common bacteria and fungi. Also, the specificity is verified every 6 months using standards containing multiple species of bacteria. The standards contain equal concentrations or varying concentrations of bacteria species. These identified compositions on the sequencer are compared to the known standard values to ensure accuracy. The varied concentration specimen is compared to the previous sequence run of the standard to verify the same organisms are in abundance and other organisms are in minor amounts.

The Next-Gen DNA Sequencing has a typical turnaround time of 3-5 business days from time of receipt of sample to our lab.

Southwest Regional PCR Laboratory DBA MicroGen DX will notify all clients of any changes to methods or procedures that may change the interpretation of data.

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