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HelpTest Code ENTP Enterovirus, Molecular Detection, PCR, Plasma
Reporting Name
Enterovirus PCR, PUseful For
Aiding in diagnosing enterovirus infections using plasma specimens
This test should not be used to screen asymptomatic patients.
Method Name
Real-Time Polymerase Chain Reaction (PCR)/RNA Probe Hybridization
Performing Laboratory
Mayo Clinic Laboratories in Rochester
Specimen 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.
Specimen Minimum Volume
0.3 mL
Specimen Stability Information
| Specimen Type | Temperature | Time | Special Container |
|---|---|---|---|
| Plasma EDTA | Refrigerated (preferred) | 7 days | |
| Frozen | 7 days |
Reject Due To
| Gross hemolysis | Reject |
Reference Values
Negative
Day(s) Performed
Monday through Sunday
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 |
Clinical Information
Enteroviruses are positive-sense RNA viruses in the Picornaviridae family. These viruses were initially classified by serotype as polioviruses (3 types), echoviruses (31 types, including types 22 and 23, which are now classified as parechoviruses), coxsackievirus A (23 types), and coxsackievirus B (6 types). However, genomic studies have demonstrated that there is significant overlap in the biological characteristics of different serotypes and, more recently, isolated enteroviruses are now named with consecutive numbers (eg, EV68, EV69).
The normal site of enterovirus replication is the gastrointestinal tract where the infection is typically subclinical. However, in a proportion of cases, the virus spreads to other organs, causing systemic manifestations, including mild respiratory disease (eg, common cold); conjunctivitis; hand, foot, and mouth disease; aseptic meningitis; myocarditis; and acute flaccid paralysis. Collectively, enteroviruses are the most common cause of upper respiratory tract disease in children. In addition, the enteroviruses are the most common cause of central nervous system (CNS) disease; they account for almost all viruses recovered in culture from spinal fluid. Differentiation of enteroviruses from other viruses and bacteria that cause CNS disease is important for the appropriate medical management of these patients.
Traditional cell culture methods require 6 days, on average, for enterovirus detection. In comparison, real-time polymerase chain reaction (PCR) allows same-day detection. Detection of enterovirus nucleic acid by PCR is also the most sensitive diagnostic method for the diagnosis of CNS infection caused by these viruses.
Interpretation
A positive result indicates the presence of enterovirus RNA in the specimen.
Cautions
A negative result does not rule out the possibility of enterovirus infection.
This assay may detect virus from a variety of specimen types in asymptomatic individuals. This assay should only be used for patients with a clinical history and symptoms consistent with enterovirus infection and must be interpreted in the context of the clinical picture.
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)