MICROBIOLOGY | Lab Services

Antimicrobial Resistance Gene Detection using qPCR

Resistance factors are run on a qualitative assay, which will report them out as present or absent.

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.

Bactrim Level 1:
Level 1 test for Bactrim is a real-time quantitative PCR assay utilizing the Roche LightCycler 480. This taqman assay uses dye labelled hydrolysis probes to detect the presence of a unique sequence in the sul I and sul II genes specific to Bactrim resistance. Sulfonamides and TMP target two enzymes, dihydropteroate synthetase or DHPS and dihydrofolate reductase or DHFR, to disrupt steps in the folic acid production cycle by inhibiting the production of dihydrofolic acid and tetrahydrofolic acid. Transferable resistance to bactrim is mediated by two drug-resistant DHPS enzymes, which are encoded in sul I and sul II genes. The accuracy, sensitivity, and specificity of the qPCR assay are 100%. The limit of detection is 0.004ng/uL.

Extended Spectrum Beta Lactamase CTX-M Level 1: 
Level 1 test for Extended Spectrum Beta Lactamase or ESBLs is a real-time quantitative PCR assay utilizing the Roche LightCycler 480. This taqman assay uses dye labelled hydrolysis probes to detect the presence of a unique sequence in the CTX-M gene specific to ESBL resistance. Extended spectrum Beta lactamases or ESBLs have resistance to penicillins, broad spectrum cephalosporins with an oxyimino side chain, such as cefotaxime, ceftriaxone, and ceftazidime, and oxyimino-monobactam aztreonam. There are 10 genes that have been documented to be associated with ESBLs, including CTX-M, SHV, TEM, PER, VEB, BES, GES, TLA, SFO and OXA; however, CTX-M makes up the largest growing group of ESBLs with the highest clinical impact. They can be found in at least 26 bacterial species and the most common are Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis. The accuracy, sensitivity, and specificity of the qPCR assay are 100%. The limit of detection is 0.0006ng/uL.