aDetection only occurs if patients are followed up
proactively from the time of exposure.
Ig, immunoglobulin; PCR, polymerase chain reaction; RT-PCR; real-time
reverse transcription PCR; SARS-CoV-2, severe acute respiratory syndrome
coronavirus 2
Real-time reverse transcriptase polymerase chain reaction
(rRT-PCR)
Real-time polymerase chain reaction (rRT-PCR) is the gold-standard
molecular technique for the detection of SARS-CoV-2 viral RNA in all
recommended samples. It targets the following sequences that code for
structural viral proteins: spike (S), membrane (M), envelope (E),
nucleocapsid (N), and RNA-dependent RNA polymerase (RdRP). Both S and N
proteins are highly immunogenic (Ravi et al., 2020). The S proteins
seems to be the major target of neutralizing antibodies for correlated
coronaviruses (Berry et al., 2010). High infectivity of SARS-CoV-2 has
compelled the CDC to publish rRT-PCR primers and probes together with
all relevant literature for public access (Khalaf et al., 2020). The
positive rate of rRT-PCR detection is dependent on the sample type, with
differences between bronchoalveolar lavage fluid (93%),
fibrobronchoscopy brush biopsy (46%), sputum (72%), nasal swabs
(63%), pharyngeal swabs (32%), feces (29%), and blood (1%) (Wang et
al., 2020). Combining nasopharyngeal and oropharyngeal swabs is now one
of the most commonly used specimen types for diagnosing COVID-19 active
infection (Lai and Lam, 2020). In September 2020, the WHO published a
guideline not recommending saliva as the only specimen type for routine
clinical diagnostics, because of the wide variation in collection
methods (World Health Organization, 2020a).
The virus can be detected at least 48 hours before the onset of symptoms
(pre-symptomatic cases), up to 12–14 days (at least 6–7 days) after
the onset of symptoms in samples from the upper respiratory tract (NP/OP
swabs), and for a median of 20 days in samples from the lower
respiratory tract, including sputum, tracheal aspirate, and
bronchoalveolar lavage (Mallett et al., 2020) (World Health Organization,
2020a) (Lippi et al., 2020).
Pooling rRT-PCR samples increases testing efficiency, which may be
particularly helpful in areas with low prevalence and few health
resources, given that only a limited number of tests are available (He
et al., 2020). The idea is to pool samples from several individuals and
test the combined sample with a single test. If the test is negative,
all subjects are negative. If the test is positive, all individuals must
be tested again to find the infected patient(s) (Food and Drug
Administration USA, 2020). The US Food and Drug Administration initially
proposed that five was the maximum number of samples to be pooled for
rRT-PCR, but other studies found that the ideal number of pooled samples
depends on the disease prevalence in the tested population (Hanel and
Thurner, 2020) (Deckert et al., 2020) (Cherif et al.,
2020). One potential constraint of pooled testing is
that the false-negative rate may increase owing to dilution of positive
samples, therefore high-sensitivity rRT-PCR tests are adequate to
minimize this limitation (Lorentzen et al., 2020). In general, the
larger the pool of specimens, the higher the likelihood of generating
false-negative results (CDC, 2020a).
As with all diagnostic tests, the predictive value of rRT-PCR depends
highly on its specificity, sensitivity, and prevalence of the disease in
the target population (CDC, 2020a) (Table 1). False-negative results may
also result from technical issues, from sampling to amplification,
including thermal inactivation (Siam et al., 2020). A confirmatory test
(e.g. repeated rRT-PCR) may be warranted if the initial results are
negative, and the clinical characteristics are suggestive of
infection (Lai and Lam, 2020) (Lorentzen et al., 2020) .