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

Ten Years' Experience Offers Speed & Accuracy

 
 

College of American Pathology (CAP) Proficiency Results

Microbial Identification to the Species Level Using NGS 2009 - 2018

Species PanelTest FrequencyNumber of Blinded Samples per TestNumber of Blinded SpecimensProficiency Score
Blinded Specimens Total 799Mean Accuracy Rate 99.4%
IDR3 Times per year for 5 years57597.3%
MRS-5 (M)3 Times per year for 10 years5150100.0%
F13 Times per year for 10 years515098.0%
D83 Times per year for 10 years515099.3%
VRE3 Times per year for 10 years240100.0%
GIP3 Times per year for 2 years530100.0%
HC-63 Times per year for 1 year515100.0%
TVAG3 Times per year for 1 year39100.0%
Alternate3 Times per year for 10 years6180100.0%

qPCR Panel Rapid Screening

The qPCR Rapid Screening 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 and methicillin.

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 their 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 analytes’ concentration in order to negate the slight loss of cells during the extraction process. Next, each initial target bacteria or fungi DNA, whose concentration was measured to obtain an initial concentration ng/uL, was diluted to obtain a six to eight 1:10 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.

Click Here for MicroGenDX qPCR Panels
Blood: Level 1 – qPCR Panel

Total 16S Bacterial Load
and Resistance Genes

Quinolone resistance
Methicillin resistance
Vancomycin resistance
Beta-lactam resistance
Carbapenem resistance
Macrolide resistance
Aminoglycoside resistance
Tetracycline resistance
Bactrim resistance
Extended Spectrum Beta Lactamase CTX-M resistance

Bacteria:
Enterococcus faecalis
Streptococcus agalactiae
(group B)
Streptococcus pyogenes
(group A)
Enterococcus faecium
Pseudomonas aeruginosa
Staphylococcus aureus
Klebsiella pneumoniae

Fungus:
Candida albicans
ENT/Pulmonary: Level 1 - qPCR Panel

Total 16S Bacterial Load
and Resistance Genes

Quinolone resistance
Methicillin resistance
Vancomycin resistance
Beta-lactam resistance
Carbapenem resistance
Macrolide resistance
Aminoglycoside resistance
Tetracycline resistance
Bactrim resistance
Extended Spectrum Beta Lactamase CTX-M resistance

Bacteria:
Haemophilus influenzae
Streptococcus agalactiae
(group B)
Streptococcus pyogenes
(group A)
Moraxella catarrhalis
Pseudomonas aeruginosa
Staphylococcus aureus
Streptococcus pneumoniae
Klebsiella pneumonia

Fungus:
Candida albicans
Gastrointestinal: Level 1 - qPCR Panel (PCR Only)

Bacteria:
Campylobacter sp (jejuni, coli, upsaliensis)
Clostridium difficile (toxin A/B)
Plesiomonas shigelloides
Salmonella sp
Yersinia enterocolitica
Vibrio sp (parahaemolyticus, vulnificus, cholerae)
Vibrio cholerae
Enteroaggregative E. coli (EAEC)
Enteroaggregative E. coli (EPEC)
Enterotoxigenic E. coli (ETEC) it/st
Shiga-like toxin producing
E. coli (STEC) stx1/stx2
E. coli O157
Shigella/Enteroinvasive
E. coli (EIEC)

Virus:
Astrovirus
Adenovirus F40/41
Rotavirus A
Norovirus GI/GII
Sapovirus (I, II, IV, V)

Parasites:
Cryptosporidium
Giardia lamblia
Entamoeba histolytica
Cyclospora cayetanensis

Fungus:
Candida albicans
Nail: Level 1 – qPCR Panel


Total 16S Bacterial Load
and Resistance Genes

Quinolone resistance
Methicillin resistance
Vancomycin resistance
Beta-lactam resistance
Carbapenem resistance
Macrolide resistance
Aminoglycoside resistance
Tetracycline resistance
Bactrim resistance
Extended Spectrum Beta Lactamase CTX-M resistance

Bacteria:
Enterococcus faecalis
Streptococcus agalactiae
(group B)
Streptococcus pyogenes
(group A)
Enterococcus faecium
Pseudomonas aeruginosa
Staphylococcus aureus

Fungus:
Candida albicans
Trichophyton rubrum
Respiratory: Level 1 – qPCR Panel (PCR Only)


Viruses:
Influenza A (H1, H1 2009, H3)
Influenza B
Adenovirus
Coronavirus (229E, OC43, NL63, HKU1)
Human Metapneumovirus
Human Rhinovirus/Enterovirus
Parainfluenza virus (1, 2, 3, 4)
Respiratory Syncytial Virus (RSV)

Bacteria:
Bordetella pertussis
Chlamydophila
pneumoniae
Mycoplasma
pneumoniae
STI – Men/Women: qPCR Panel (PCR Only)

Chlamydia trachomatis
Mycoplasma genitalium
Neisseria gonorrhoeae
Trichomonas vaginalis
Urine/Semen/Rectal: Level 1 - qPCR Panel


Total 16S Bacterial Load
and Resistance Genes

Quinolone resistance
Methicillin resistance
Vancomycin resistance
Beta-lactam resistance
Carbapenem resistance
Macrolide resistance
Aminoglycoside resistance
Tetracycline resistance
Bactrim resistance
Extended Spectrum Beta Lactamase CTX-M resistance

Bacteria:
Pseudomonas aeruginosa
Enterococcus faecalis
Streptococcus agalactiae
Klebsiella pneumoniae
Staphylococcus aureus
Proteus mirabilis
E coli
Gardnerella vaginalis
Mobiluncus curtisii
Mobiluncus mulieris
Staphylococcus saprophyticus
Prevotella bivia
Ureaplasma urealyticum
Ureaplasma parvum
Lactobacillus gasseri
Lactobacillus -
crispatus/acidophilus
Mycoplasma hominis

Fungus:
Candida albicans
UTI/STI Combined: Level 1 - qPCR Panel


Total 16S Bacterial Load
and Resistance Genes

Quinolone resistance
Methicillin resistance
Vancomycin resistance
Beta-lactam resistance
Carbapenem resistance
Macrolide resistance
Aminoglycoside resistance
Tetracycline resistance
Bactrim resistance
Extended Spectrum Beta Lactamase CTX-M resistance

Bacteria:
Pseudomonas aeruginosa
Enterococcus faecalis
Streptococcus agalactiae
Klebsiella pneumoniae
Staphylococcus aureus
Proteus mirabilis
E coli
Gardnerella vaginalis
Mobiluncus curtisii
Mobiluncus mulieris
Staphylococcus saprophyticus
Prevotella bivia
Ureaplasma urealyticum
Ureaplasma parvum
Lactobacillus gasseri
Lactobacillus -
crispatus/acidophilus
Mycoplasma hominis

Fungus:
Candida albicans

STIs:
Chlamydia trachomatis
Mycoplasma genitalium
Neisseria gonorrhoeae
Trichomonas vaginalis
Vaginal: Level 1 - qPCR Panel


Total 16S Bacterial Load
and Resistance Genes

Quinolone resistance
Methicillin resistance
Vancomycin resistance
Beta-lactam resistance
Carbapenem resistance
Macrolide resistance
Aminoglycoside resistance
Tetracycline resistance
Bactrim resistance
Extended Spectrum Beta Lactamase CTX-M resistance

Bacteria:
Streptococcus agalactiae
(group B)
Gardnerella vaginalis
Mobiluncus curtisii
Mobiluncus mulieris
Staphylococcus saprophyticus
Prevotella bivia
Ureaplasma urealyticum
Ureaplasma parvum
Lactobacillus gasseri
Lactobacillus crispatus/
acidophilus
Mycoplasma hominis

Fungus:
Candida albicans
Vaginal Combined/STI: qPCR Panel


Total 16S Bacterial Load
and Resistance Genes

Quinolone resistance
Methicillin resistance
Vancomycin resistance
Beta-lactam resistance
Carbapenem resistance
Macrolide resistance
Aminoglycoside resistance
Tetracycline resistance
Bactrim resistance
Extended Spectrum Beta Lactamase CTX-M resistance

Bacteria:
Streptococcus agalactiae
(group B)
Gardnerella vaginalis
Mobiluncus curtisii
Mobiluncus mulieris
Staphylococcus saprophyticus
Prevotella bivia
Ureaplasma urealyticum
Ureaplasma parvum
Lactobacillus gasseri
Lactobacillus crispatus/
acidophilus
Mycoplasma hominis

Fungus:
Candida albicans

STIs:
Chlamydia trachomatis
Mycoplasma genitalium
Neisseria gonorrhoeae
Trichomonas vaginalis
Wound/Ortho: Level 1 – qPCR Panel


Total 16S Bacterial Load
and Resistance Genes

Quinolone resistance
Methicillin resistance
Vancomycin resistance
Beta-lactam resistance
Carbapenem resistance
Macrolide resistance
Aminoglycoside resistance
Tetracycline resistance
Bactrim resistance
Extended Spectrum Beta Lactamase CTX-M resistance

Bacteria:
Enterococcus faecalis
Streptococcus agalactiae
(group B)
Streptococcus pyogenes
(group A)
Enterococcus faecium
Pseudomonas aeruginosa
Staphylococcus aureus
Klebsiella pneumoniae
Proprionibacterium acnes*
(*only in ortho panel)

Fungus:
Candida albicans

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

The sensitivity and specificity are 100% on E. faecalis, E. faecium, K. pneumoniae, P. aeruginosa, S. aureus, S. pyogenes, vanA, C. albicans, M. catarrhalis, S. pneumoniae, E. coli, P. mirabilis, T. rubrum, beta-lactam, carbapenem, macrolide, aminoglycoside, tetracycline, quinolone, C. trachomatis, G. vaginalis, L. crispatus/acidophilus, L. gasseri, M. curtisii, M. genitalium, M. hominis, S. saprophyticus, and U. urealyticum. 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%. The sensitivity and specificity for M. mulieris are 100% and 98.6%. The sensitivity and specificity for N. gonorrhoeae and T. vaginalis are 100% and 99.3%. The sensitivity and specificity for P. bivia and U. parvum are 100% and 99.2%.

The accuracy of the above assays is 100% on all except mecA, 16S, H. influenzae, M. mulieris, N. gonorrhoeae, T. vaginalis, P. bivia, and U. parvum. The accuracy is 98.9%, 99.6%, 99.6%, 98.7%, 99.4%, 99.3%, 99.4%, and 99.3% for mecA, 16S, H. influenzae, M. mulieris, N. gonorrhoeae, T. vaginalis, P. bivia, and U. parvum, respectively.

Southwest Regional PCR 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.

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.

Next-Gen Sequencing Comprehensive DNA Test

The Comprehensive Sequencing 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 Illumina MiSeq 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 MiSeq sequencer by a "tag," a unique identifying sequence attached to the forward and reverse primers implemented when the targeted sequence is amplified using polymerase chain reaction or PCR. The PCR contains a 5 minute denaturation step at 95˚C, 35 cycles of 94˚C for 30 seconds, 52˚C for 40 seconds, and 72˚C for 60 seconds, and a final extension step of 72˚C for 10 minutes. After the sample DNA has been amplified and each sample has its own unique sequence tag, the sample DNA can be pooled together based on amplification strength for downstream applications. Purification of the pooled DNA will be done by removing small fragments using both Agencourt Ampure beads and Qiagen Minelute kit. The DNA is quantified and prepared for sequencing. Finally, the DNA library is run on the MiSeq sequencer.

The Illumina MiSeq sequencer uses bridge PCR to generate clusters for each of the specimens present in the DNA library and amplify their sequences. Once those clusters are formed, sequencing begins through the incorporation of fluorescently labelled nucleotides with reverse terminators and those fluorescent signals are captured by the MiSeq’s camera. The reverse terminator attached to each nucleotide allows the addition of just one nucleotide per cycle and removes the problem with homopolymers that is suffered from other sequencing platforms. In addition, the MiSeq sequencer reads both strands, forward and reverse, of DNA, which provides an increase in accuracy and a decrease in error rate.

The sequencing reads are analyzed for quality and length during the data analysis pipeline. The data analysis pipeline consists of two major stages, the denoising and chimera detection stage and the microbial diversity analysis stage. During the denoising and chimera detection stage, denoising is performed using various techniques to remove short sequences, singleton sequences, and noisy reads. With the low quality reads removed, chimera detection is performed to aid in the removal of chimeric sequences. Any read that fall below the quality score or quality metric or appropriate length are discarded due to the poor quality. Only the high-quality reads will be interpreted and reported to physicians.

The high-quality sequencing reads that are left are compared to our internally maintained and curated database. There are many publicly available databases that allow researchers from all over the world to collaborate new genomes, new strains, and reclassification of new species. With this large influx of ever-growing knowledge stems a new problem, how to verify and check the information. The decision to move away from a publicly available database, like NCBI and GreenGenes, to one that was curated and maintained in house solved this problem. In order for the sequences in the available databases to be incorporated into the in-house database, they must go through 3 stages of quality checking, including quality standard, taxonomy, and accuracy stages. In addition, the in-house database contains strain and species’ sequences from MicroGen’s 10 years of clinical diagnostic experience. The percentage of each organism will be individually analyzed for each sample providing relative abundance information within and among the individual samples based upon relative numbers of reads within each.

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 1000 copies of each species. Each organism is reported as a relative abundance in the sample.

The Comprehensive Sequencing test has a turnaround time of fourteen days (including weekends), but it is most commonly completed between 3-7 business days.

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

MicroGenDX Curated Species Database for Accurate NGS Results

The database at MicroGenDX, one of the first NGS companies in the U.S.A, is heavily curated before being used for patient testing. For sequences to be entered into this database, they must go through several stages of quality verification. First, the sequences must cover genes of interest. Then, the sequences must be annotated in the proper format. If the annotation is incomplete, then they will not be incorporated for verification. Next, the sequences will be tested for accuracy by verifying that the sequences are attributed to the correct species. Any new sequence such as the ones called novel but match a current strain (i.e., incorrectly annotated), will be found during the accuracy check and incorporated under the proper name. The sequences are then run against a test set of species that have been sequenced in house from ATCC standards to ensure that the proper identification of these known positives has not been skewed. Lastly, any taxonomy changes to previous or current species are updated in the database. This curation occurs every 6 months.

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