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HelpTest Code LAB1231740 Enterovirus, Molecular Detection, PCR, Plasma
Performing Laboratory
Mayo Clinic Laboratories in Rochester
Reporting Name
Enterovirus PCR, PSpecimen Type
Plasma EDTAOrdering Guidance
This test will detect enterovirus but will not differentiate viruses in this family or provide serotyping information.
Specimen Required
Submit a raw clinical sample (not a culture isolate) for enterovirus polymerase chain reaction .
Collection Container/Tube: Lavender top (EDTA)
Submission Container/Tube: Screw-capped, sterile container
Specimen Volume: 1 mL
Collection Instructions: Centrifuge blood collection tube and aliquot plasma into plastic vial.
Reject Due To
| Gross hemolysis | Reject |
Specimen Stability Information
| Specimen Type | Temperature | Time |
|---|---|---|
| Plasma EDTA | Refrigerated (preferred) | 7 days |
| Frozen | 7 days |
Specimen Minimum Volume
0.3 mL
Day(s) Performed
Monday through Sunday
Specimen Retention Time
1 weekReport Available
Same day/1 to 5 daysReference Values
Negative
Useful For
Aiding in diagnosing enterovirus infections using plasma specimens
This test should not be used to screen asymptomatic patients.
CPT Code Information
87498
LOINC Code Information
| Test ID | Test Order Name | Order LOINC Value |
|---|---|---|
| ENTP | Enterovirus PCR, P | 97151-5 |
| Result ID | Test Result Name | Result LOINC Value |
|---|---|---|
| 56068 | Enterovirus PCR, P | 97151-5 |
Method Description
For this real-time reverse-transcription laboratory-developed polymerase chain reaction (PCR) assay, viral nucleic acid is extracted from specimens using the MagNA Pure automated instrument (Roche Applied Science), followed by amplification and detection on the Roche LightCycler 2.0 instrument. This PCR assay has been optimized to detect a target sequence in the polyprotein region. Primers amplify a 193 base-pair product.
Enterovirus genomic RNA is first transcribed to complementary DNA (cDNA) by reverse transcriptase, followed by amplification of the cDNA product. The LightCycler instrument can rapidly (30-40 minutes) detect amplicon development through stringent air-controlled temperature cycling in capillary cuvettes. The detection of amplified products is based on the fluorescence resonance energy transfer (FRET) principle. For FRET product detection, a hybridization probe with a donor fluorophore, fluorescein, on the 3'-end is excited by an external light source and emits light that is absorbed by a second hybridization probe with an acceptor fluorophore, LC-Red 640, at the 5'-end. The acceptor fluorophore then emits light of a different wavelength that can be measured with a signal proportional to the amount of specific PCR product. FRET (with subsequent production of a detectable fluorescent signal) only occurs when the probes have specifically annealed to the target sequence of the amplicon.
Melting curve analysis is performed following PCR amplification and is the detection phase of the assay, since it offers greater sensitivity than the amplification phase and maintains high specificity.
The melting phase of the assay occurs as follows:
Starting at 45° C, which allows the probes to bind to the amplified product, the temperature in the thermal chamber is then slowly raised to 80° C and the fluorescence measured at frequent intervals to determine the point where half of the fluorescence is lost as the probes are denatured (ie, "melt") off of the target. This is called the melting temperature (Tm) of that virus. Analysis of the PCR amplification and probe melting curves is accomplished through the use of LightCycler software.(Bernard PS, Reiser A, Pritham GH: Mutation detection by fluorescent hybridization probe melting curves. In: Meuer S, Wittwer C, Nakagawara K, eds. Rapid Cycle Real-Time PCR Methods and Applications. Springer; 2012:11-20)
Supportive Data
Accuracy/Diagnostic Sensitivity and Specificity:
Mayo Clinic Laboratories compared the generic detection of enteroviruses from spinal fluid by conventional tube cell culture (MCR-5) and by LightCycler polymerase chain reaction (PCR). Of 715 specimens tested, enteroviruses were detected in 65 (9%) by conventional cell culture and 82 (11%) by LightCycler PCR. Twenty-two of 82 (27%) were exclusively positive by PCR, whereas only 5 of 65 (8%) were exclusively positive by conventional cell cultures.
Supplemental Data (Spiking Studies):
To supplement the data above, 30 or more negative specimens of each specimen type (cerebrospinal fluid/sterile body fluid, dermal/ocular/rectal swabs, plasma, and upper and lower respiratory specimens) were spiked with enterovirus culture control at approximately 10 to 50 targets/mcL (the approximate limit of detection). The spiked specimens were run in a blinded manner along with negative (non-spiked) specimens of each specimen type. Of the spiked specimens, 97% to 100% were positive, and 100% of the non-spiked specimens were negative. A total of 489 spiked and non-spiked specimens were tested.
Assay Inclusivity:
The assay detected all 64 members of an enterovirus panel, consisting of coxsackieviruses, polio viruses, echoviruses, and other enteroviruses.
Analytical Specificity/Limit of Detection:
The lower limit of detection of this assay is approximately 10 to 50 RNA target copies/mcL. This was confirmed in all specimen types accepted for this assay.
Specificity:
The assay did not crossreact with a specificity panel containing other RNA-containing viruses (rhinovirus; reovirus; influenza virus, types A and B; respiratory syncytial virus; and parainfluenza virus) and DNA-containing viruses (herpes simplex, Epstein-Barr virus, varicella-zoster virus, and cytomegalovirus).
Reportable Range:
This is a qualitative assay and results are reported as either negative or positive for targeted enterovirus RNA.
Forms
If not ordering electronically, complete, print, and send a Microbiology Test Request (T244) with the specimen.