Molecular methods
The aforementioned traditional methods have been the
cornerstone for diagnosis used by virology laboratories around
the world. The introduction of nucleic acid amplification tests
(NAATs) for respiratory viruses starting in the late 1980s herald-
ed a new era in diagnosing respiratory virus infections. The first
NAAT for respiratory viruses was developed for influenza and
used a nucleic acid amplification method called polymerase
chain reaction (PCR), developed in 1983 by Kary B. Mullis, who
was later awarded the Nobel Prize in Chemistry in 1993. Within
a decade, NAATs were developed for all of the respiratory virus-
es, and most used PCR; however, other amplification schemes,
such as nucleic acid–sequence-based amplification (NASBA),
strand displacement amplification (SDA), transcription-mediat-
ed amplification (TMA), and loop-mediated isothermal ampli-
fication (LAMP), have also been used. For all NAATs, the total
nucleic acid is first extracted from the respiratory tract speci-
men using a variety of methods, and the viral ribonucleic acid
(RNA) is copied into a complementary deoxyribonucleic acid
(cDNA) using an enzyme called reverse transcriptase. The cDNA
is then amplified by PCR using virus-specific oligonucleotide
primers, resulting in a billion copies of DNA that can be easily
detected by a variety of common laboratory methods. Follow-
ing the emergence of new human respiratory viruses in the
twenty-first century, there has been a need for new diagnostic
tests to detect these viral pathogens, and NAAT filled this need.
Early comparisons of molecular and traditional methods clearly
indicated that the molecular methods were more sensitive than
the traditional methods, often diagnosing up to 30% additional
infected patients. Molecular testing methods also provided
test results for clinicians often within 1 day (as compared with
2–5 days needed for traditional methods), thus improving their
management of patients.
The next major advance in diagnostics was the development
of multiplex PCR (M-PCR) for the detection of several different
viruses in a single test. M-PCR uses multiple oligonucleotide
primers, with one pair for each virus to be detected. Because
M-PCR will detect several different viruses, a method is required
to identify which virus is present in the specimen. This is done
using a microarray (a collection of several different DNA
oligonucleotides) that is either spotted onto microscope slides
or cartridges (gene chips) or immobilized onto microspheres
(microfluidic arrays) that are each uniquely labeled using a
mixture of fluorescent dyes and identified by lasers. Each
element in the array (a spot or microsphere) consists of a
unique oligonucleotide (representing a unique virus) that will
bind individual PCR products for each virus type or subtype.
A positive specimen will generate an amplification product
that is hybridized to one of the elements of the microarray and
detected by a laser. One M-PCR called the xTAG respiratory viral
panel (RVP) is used as an in vitro diagnostic device. This test
was designed to identify 20 different respiratory virus types and
subtypes, and it uses more than 30 primers for target amplifica-
tion and identification. Although M-PCR tests are slightly more
expensive than single NAATs, they have the advantage of being
able to detect many different viruses in a single test, as well as
being able to detect dual infections occurring in about 10% of
patients and even triple infections that are not often seen with
traditional methods. NAATs using M-PCR and microarray tech-
nologies offer unprecedented power for the laboratory, and are
well on the way to becoming the pillars of diagnostic virology
for the present century.
Detection of Respiratory Viruses (continued)
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