#PCR
RT-PCR FOR DETECTION OF CORONAThe COVID-19 RT-PCR test is a real-time reverse transcription polymerase chain
reaction (rRT-PCR) test for the qualitative detection of nucleic acid from SARS-CoV-2
in upper and lower respiratory specimens (such as nasal, nasopharyngeal or
oropharyngeal swabs, sputum, lower respiratory tract aspirates, bronchoalveolar lavage,
and nasopharyngeal wash/aspirate or nasal aspirate) collected from individuals suspected
of COVID-19 by their healthcare provider.
This test is also for use with the Pixel by LabCorp™ COVID-19 test home collection kit
to self-collect nasal swab specimens at home by individuals when determined by a
healthcare provider to be appropriate based on results of a COVID-19 questionnaire.
Testing is limited to the Center for Esoteric Testing, Burlington, NC, or other laboratories
designated by LabCorp that are also certified under the Clinical Laboratory Improvement
Amendments of 1988 (CLIA), 42 U.S.C. §263a, to perform high complexity tests.
Results are for the identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is
generally detectable in respiratory specimens during the acute phase of infection. Positive
results are indicative of the presence of SARS-CoV-2 RNA; clinical correlation with
patient history and other diagnostic information is necessary to determine patient
infection status. Positive results do not rule out bacterial infection or co-infection with
other viruses. The agent detected may not be the definite cause of disease. Laboratories
within the United States and its territories are required to report all positive results to the
appropriate public health authorities.Negative results do not preclude SARS-CoV-2 infection and should not be used as thesole basis for patient management decisions. Negative results must be combined withclinical observations, patient history, and epidemiological information.
The COVID-19 RT-PCR Test is a real-time reverse transcription polymerase chain
reaction (rRT -PCR) test. The test can be run in a singleplex format (three individual
assays) or multiplexed into a single reaction and amplification set up. In a singleplex
format, the test uses three primer and probe sets to detect three regions in the SARSCoV-2 nucleocapsid (N) gene and one primer and probe set to detect human RNase P
(RP) in a clinical sample. When multiplexed into a single reaction, the test uses two
primer and probe sets to detect two regions in the SARS-CoV-2 N gene and one primer
and probe set to detect RP. RNA isolated from upper and lower respiratory specimens
(such as nasal, nasopharyngeal or oropharyngeal swabs, sputum, lower respiratory tract
aspirates, bronchoalveolar lavage, and nasopharyngeal wash/aspirate or nasal aspirate) is
reverse transcribed to cDNA and subsequently amplified using Applied Biosystems
QuantStudio7 Flex (QS7) instrument with software version 1.3. During the amplification
process, the probe anneals to a specific target sequence located between the forward and
reverse primers. During the extension phase of the PCR cycle, the 5’ nuclease activity of
Taq polymerase degrades the bound probe, causing the reporter dye to separate from the
quencher dye, generating a fluorescent signal. Fluorescence intensity is monitored at each
PCR cycle by QS7.Fluorescence spectroscopy analyzes fluorescence from a molecule based on its fluorescent properties.Fluorescence is a type of luminescence caused by photons exciting a molecule, raising it to an electronic excited state.Fluorescence spectroscopy uses a beam of light that excites the electrons in molecules of certain compounds, and causes them to emit light. That light is directed towards a filter and onto a detector for measurement and entification of the molecule or changes in the molecule.Introduction to Steady State and Time Resolved Fluorescence SpectroscopyThe term fluorescence refers to one type of luminescence. Luminescence, broadly defined, is light emission from a molecule. There are several types of luminescence.Photoluminescence is when light energy, or photons, stimulate the emission of a photon.Chemiluminescence, is defined as when chemical energy stimulates the emission of a photon, and this includes bioluminescence, as seen in fire flies and many forms of sea life.
Electroluminescence, is when electrical energy or a strong electric field, stimulates the emission of a photon, such as in some lighting applications.Fluorescence, specifically, is a type of photoluminescence where light raises an electron to an excited state. The excited state undergoes rapid thermal energy loss to the environment through vibrations, and then a photon is emitted from the lowest-lying singlet excited state. This process of photon emission competes for other non-radiative processes including energy transfer and heat loss.When the term “fluorescence” is used, the same methods of measurement can typically be applied to any of the above categories of luminescence. luorometry or spectrofluorometry,