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Microbial identification assays
qPCR assays for rapid, sensitive microbial and viral detection
Save time spent designing primers and probes by using predesigned assay sets. The following
- C. difficile toxin
- Influenza A, B/RSV
- HSV-1/HSV-2/VZV
- PivNG V2
Primers and probes are HPLC purified and verified by ESI mass spectrometry, delivering batch-to-batch consistency to provide confidence in your data.
Product details
Assay validation
The primers and probes were tested using the Roche cobas omni Utility Channel Tool v3.0 and the cobas omni Utility Channel Reagent Kit, using the parameters shown in Tables 1 and 2.
Table 1. Experimental conditions.
| C. difficile toxin | HSV-1 / HSV-2 / VZV | Influenza A / Influenza B / RSV | PivNG V2 | |
|---|---|---|---|---|
| Channel number | Channel 2 (C. diff toxin) | Channel 2 (HSV-1) Channel 4 (HSV-2) Channel 3 (VZV) | Channel 2 (Influenza A) Channel 3 (Influenza B) Channel 4 (RSV) | Channel 4 (NG) |
| Primer/probe set volume | 480 µL | 480 µL | 480 µL | 480 µL |
| MMx-2 | 8 mL | 8 mL | 8 mL | 8 mL |
| 7 mL of the primer-probe and MMx-2 mix transferred to the cobas omni Utility Channel Reagent cassette | ||||
| Sample type selected | Simple sample | Simple sample | Swab sample | Simple sample or swab sample |
| Sample input volume | 200 µL | 200 µL (CSF) 500 µL (plasma and swab) | 400 µL | 850 µL (simple) 400 µL (swab) |
| RFI min | 1.5 (C. diff toxin) | 1.6 (HSV-1) 1.5 (HSV-2) 1.8 (VZV) | 1.3 (Influenza A) 2 (Influenza B) 1.2 (RSV) | 2.5 (NG) 2.5 (IC) |
Table 2. Cycling conditions.*
| UNG incubation | Pre-PCR step | 1st measurement | 2nd measurement | Cooling | |
|---|---|---|---|---|---|
| No. of cycles | Predefined | 1 | 5 | 45 | Predefined |
| No. of steps | 3 | 2 | 2 | ||
| Temperature | 55°C; 60°C; 65°C | 95°C; 55°C | 91°C; 58°C | ||
| Hold time | 120 s; 360 s; 240 s | 5 s; 30 s | 5 s; 25 s | ||
| Data acquisition | None | End of each cycle | End of each cycle |
* Default PCR profile for the cobas omni Utility Channel provided by the Utility Channel software.
Specificity information
Tables 3–5 provide information on the organisms the C. diff, HSV/VZV, and Influenza qPCR Assays can specifically identify.
Table 3. Assays for Clostridium
| C. difficile | ||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Quality control panel (all panel members correctly identified) | Qnostics C. difficile Panel (March 2016), including C. difficile ribotypes 027 (Toxin: A+/B+) and 017 (Toxin: A-/B+) ZeptoMetrix NATrol™ C. difficile Verification Panel (including ribotypes 002, 017, 078, 027-1, and 027-2) | |||||||||||||||
| Culture fluids (all organisms correctly identified) | None | |||||||||||||||
| No cross-reactivity (none of the organisms generated |
|
Table 4. Assays for herpes simplex virus, types 1 and 2 (HSV-1, HSV-2), Varicella-zoster virus (VZV).
| Herpes simplex virus, types 1 and 2 (HSV-1, HSV-2), Varicella-zoster virus (VZV) | ||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Quality control panel (all panel members correctly identified) | INSTAND e.V., Virus Genome Detection—Herpes Simplex Virus Type 1/Type 2 (363) ( June, 2014) | |||||||||||||||||||||||||||||
| Culture fluids (all organisms correctly identified) |
| |||||||||||||||||||||||||||||
| No cross-reactivity (none of the organisms generated false positive results) |
|
Table 5. Assays for Influenza A, Influenza B, and Respiratory syncytial virus (RSV).
| Influenza A, Influenza B, and RSV | |||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Quality control panel (all panel members correctly identified) | Qnostics Influenza A/B panels (including H1N1, H3N2, and H5N1) and RSV panel (RSV-A and RSV-B) | ||||||||||||||||||||||||||||||
| Culture fluids (all organisms correctly identified) |
| ||||||||||||||||||||||||||||||
| No cross-reactivity (none of the organisms generated false positive results) |
|
An in silico assessment was also performed, comparing the Influenza/RSV qPCR Assay primers and probes against 1777 Human Flu A strains and 913 Human Flu B strains.
With respect to Flu A, 98.2% of the primers were perfect matches, while the remaining 1.8% of the primers had only 1 mismatch. 42.8% of the Flu A probes were perfect matches, while 56.7% of the probes had 1 or 2 mismatches, and the remaining 0.6 % had 3 mismatches. See the NCBI and CDC Flu A primer and probe alignment data for the individual targets tested here.
With respect to Flu B, 94.3% of the primers were perfect matches, while the remaining 5.6% of the primers had only 1 or 2 mismatches. 69.4% of the Flu B probes were perfect matches, while the remaining 30.6% of the probes had 1 or 2 mismatches. See the NCBI and CDC Flu B primer and probe alignment data for the individual targets tested here.
In silico data is not intended to be, and should not be interpreted as being, in any way predictive of assay performance.
The strains compared were from the following sources:
- The CDC-2018 viral strains used to develop the current viral vaccine; and
- The NCBI database (https://www.ncbi.nlm.nih.gov/genomes/FLU), accessed on 05/11/2018 for all Human Influenza A sequences collected between January 1, 2008 and November 5, 2018.
Table 6. PivNG V2
| Quality control panel (all panel members correctly identified) | ZeptoMetrix NATrol™ CT/NG Verification Panel (NATCT/NGP-C). |
|---|---|
| Culture fluids (all organisms correctly identified) | None |
| No cross-reactivity (none of the organisms generated false positive results) | Neisseria cinerea Neisseria denitrificans Neisseria elongata Neisseria elongata subsp. elongata Neisseria elongata subsp. niroreducans Neisseria flava Neisseria flavescens Neisseria kochi Neisseria lactamica Neisseria macacae Neisseria meningitidis Serogroup A Neisseria meningitidis Serogroup B Neisseria meningitidis Serogroup C Neisseria meningitidis Serogroup D Neisseria meningitidis Serogroup W135 Neisseria meningitidis Serogroup Y Neisseria mucosa Neisseria perflava Neisseria polysaccharea Neisseria sicca Neisseria subflava Neisseria weaveri |
Legal and regulatory information
This product and its use is the subject of one or more U.S. patent, and/or other pending U.S. and foreign patent applications owned by IDT. This includes patents and patent applications of IDT related to the right to use oligonucleotide probes labeled with Iowa Black® FQ, Iowa Black RQ, TAO™, and/or ZEN™ quenchers. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research and diagnostics for human healthcare conducted by the buyer. The buyer cannot sell or otherwise transfer this product or its components for a third party.
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