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Test Code ENTP Enterovirus, Molecular Detection, PCR, Plasma

Reporting Name

Enterovirus PCR, P

Useful For

Aids in diagnosing enterovirus infections

Performing Laboratory

Mayo Clinic Laboratories in Rochester

Specimen Type

Plasma EDTA


Specimen Required


Submit a raw clinical sample (not a culture isolate) for enterovirus PCR. This test will detect enterovirus, but will not differentiate viruses in this family or provide serotyping information.

 

Collection Container/Tube: Lavender top (EDTA)

Submission Container/Tube: Screw-capped, sterile container

Specimen Volume: 1 mL

Collection Instructions: Spin down promptly.


Specimen Minimum Volume

0.3 mL

Specimen Stability Information

Specimen Type Temperature Time Special Container
Plasma EDTA Refrigerated (preferred) 7 days
  Frozen  7 days

Reference Values

Negative

Day(s) and Time(s) Performed

Monday through Sunday; Varies

Test Classification

This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the U.S. Food and Drug Administration.

CPT Code Information

87498

LOINC Code Information

Test ID Test Order Name Order LOINC Value
ENTP Enterovirus PCR, P 29591-5

 

Result ID Test Result Name Result LOINC Value
56068 Enterovirus PCR, P 29591-5

Clinical Reference

1. Enterovirus surveillance-United States, 1970-2005. MMWR Morb Mortal Wkly Rep Sept 15 2006;55(SS08):1-20

2. Foray S, Pailloud F, Thouvenot D, et al: Evaluation of combining upper respiratory tract swab samples with cerebrospinal fluid examination for the diagnosis of enteroviral meningitis in children. J Med Virology 1999;57(2):193-197

3. Furione M, Zavattoni M, Gatti M, et al: Rapid detection of enteroviral RNA in cerebrospinal fluid (CSF) from patients with aseptic meningitis by reverse transcription-nested polymerase chain reaction. New Microbiol 1998;21(4):343-351

Method Description

For this real-time reverse-transcription laboratory-developed 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-bp product.

 

Enterovirus genomic RNA is first transcribed to 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 a light of a different wavelength that can be measured with a signal that is 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° Celsius, which allows the probes to bind to the amplified product, the temperature in the thermal chamber is then slowly raised to 80° Celsius and the fluorescence is 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.(Cockerill FR III, Uhl JR: Applications and challenges of real-time PCR for the clinical microbiology laboratory. In Rapid Cycle Real-Time PCR Methods and Applications. Edited by U Reischl, et al. Germany, Springer, 2002, pp 3-30)

Analytic Time

Same day/1 day

Reject Due To

Gross hemolysis Reject

Method Name

Real-Time Polymerase Chain Reaction (PCR)/RNA Probe Hybridization

Forms

If not ordering electronically, complete, print, and send a Microbiology Test Request (T244) with the specimen.