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Coronavirus-SARS-CoV-2: Biology and Problems in rRT-PCR Detection

In December 2019, a severe respiratory disease appeared in China, which is now named coronavirus disease 2019 (COVID-19) (World Health Organization, 2020a). The World Health Organization has declared the COVID-19 outbreak a pandemic. This disease has spread to 215 countries, infecting more than six million people and causing more than 370 thousand deaths as of May 31st, 2020 (Worldometer, 2020). COVID-19 is a disease caused by Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) infection with symptoms of cough, fever, fatigue, headache, and shortness of breath (Guan et al., 2020; Wang et al., 2020). The diagnosis of COVID -19 is made through observing the clinical manifestations that arise and the diagnostic examination of SARS-CoV-2. The SARS-CoV-2 diagnostic tests are carried out through Reverse Transcription polymerase chain reaction (RT-PCR) testing as a method to confirm the diagnosis of cases of COVID -19 according to WHO guidelines (World Health Organization, 2020b). In this paper, we discuss the biology of SARS-CoV-2 and the principles of RT-PCR as the basis to understand the method of detection of SARSCoV-2 and problems that may occur using this detection method.


INTRODUCTION
In December 2019, a severe respiratory disease appeared in China, which is now named coronavirus disease 2019   (World Health Organization, 2020a). The World Health Organization has declared the COVID-19 outbreak a pandemic. This disease has spread to 215 countries, infecting more than six million people and causing more than 370 thousand deaths as of May 31 st , 2020 (Worldometer, 2020). COVID-19 is a disease caused by Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) infection with symptoms of cough, fever, fatigue, headache, and shortness of breath (Guan et al., 2020;Wang et al., 2020).
The diagnosis of COVID -19 is made through observing the clinical manifestations that arise and the diagnostic examination of SARS-CoV-2. The SARS-CoV-2 diagnostic tests are carried out through Reverse Transcription polymerase chain reaction (RT-PCR) testing as a method to confirm the diagnosis of cases of COVID -19 according to WHO guidelines (World Health Organization, 2020b). In this paper, we discuss the biology of SARS-CoV-2 and the principles of RT-PCR as characteristics and positive-sense RNA as genetic material. The length of the coronavirus genome is about 26,4-31,7 kb. Coronaviridae and Roniviridae family are the largest RNA virus among other viruses (King et al., 2011). The SARS-CoV-2 genome is 29.9 kb (Forster et al., 2020).
As a member of Nidovirales, coronavirus is classified into the family of Coronaviridae which usually has three or four envelope proteins. Viruses that belong to   (King et al., 2011) Coronavirus has a few E proteins on the outer layer of its envelope. It is a pentameric integral membrane protein that facilitates the movement of molecules passing through the ion channel and/or viroporin. Even though there is only a small number of this protein, it was identified as an essential element of virion morphogenesis and a virulent factor of Severe Acute Respiratory Syndrome Virus (SARS)-CoV (King et al., 2011). Wan et al. (2020) reported that SARS-CoV-2 and b bat SARS-like coronavirus were in the same b genus.
Both were also originated from the same ancestral line.
Human SARS-CoV and bat SARS-CoV can infect a target cell through angiotensin-converting enzyme 2 (ACE2) receptor. Alignment of Receptor-Binding Domain (RBD) of SARS-CoV and SARS-CoV-2 showed that SARS-CoV-2 RBD had a mutation on 105 residues. It was predicted that the mutation can enhance the affinity of SARS-CoV-2 RBD to the ACE2 receptor (Wan et al., 2020).
Spike glycoprotein (S) is one of the targets of T cell response in the immune system. It acts as an inducer in the neutralization of virus infectivity by antibodies (King et al., 2011). The S protein also promotes the binding of the virus envelope to the ACE2 receptor and the entry of the virus into the cell target (Wan et al., 2020). kidney (Hamming et al., 2004). Despite ACE2 proteins are scattered all over endothelial and smooth muscle cells, not every organ is infected by a coronavirus. SARS-CoV-2 may need a co-receptor as in found in HIV. The HIV requires surface receptor CD4 and chemokine coreceptor for infecting and entering the cell target (Zhang et al., 1996).

POLYMERASE CHAIN REACTION
Polymerase Chain Reaction (PCR) is a DNA amplification technique that was invented by Karl Mullis in 1984(Mullis, 1987. This technology is commonly used for pathogen identification, forensic analysis (Schaad & Frederick, 2002), gene identification, and genetic engineering (Garibyan & Avashia, 2013 Primers are short pieces of single-stranded DNA that can be designed for restricting the segment of DNA target (Schaad & Frederick, 2002). In the process of amplification, the sample is used in a small amount.
Therefore, PCR becomes a sensitive technique for DNA identification (Garibyan & Avashia, 2013   b These E-gene assays are specific for bat (-related) beta coronaviruses. They detect both SARS-CoV-1 and -2. c Shipment is performed at room temperature. d According to the manufacturer, CE-IVD certification will be applied shortly.

RT-PCR KITS
All RT-PCR kits performed splendid results with PCR   (Hindson, 2020;Lo et al., 2020;Wölfel et al., 2020). The specimen is collected as soon as possible in the initial onset (Center for Disease Control and Prevention, 2020b). Research showed that the viral loads in throat swab and sputum peaked in 5-6 days after symptom onset (Pan et al., 2020). Another research indicated that viral load was higher in the nose (NP and OP swabs) than in the throat (Zou et al., 2020).

RT-PCR TEST SAMPLES
The use of NP swabs as a specimen is preferred over OP swabs due to higher detection rates. This is consistent with research that demonstrates early viral development in the nasal/NP . Sampling techniques through swabs must be performed at the right anatomy and time so that negative-false can be avoided. Besides, only synthetic fiber swabs with plastic or wire shafts are used (Center for Disease Control and Prevention, 2020b).
In NP swabs, mini tip swabs are inserted into the nose parallel to the palate until they reach the tip, namely the nasopharynx. Gently rub and roll the swab and wait a few seconds to absorb the secretion. Then slowly remove the swab while rotating it. The technique for collecting specimens in the form of an NP swab is illustrated in  hours. If a delay in testing or shipping is expected, store specimens at -70°C or below (Center for Disease Control and Prevention, 2020b). Complete information about specimen collection and storage is in Table II. Low viral load (World Health Organization, 2020b) is one of the things that can negatively impact the results of the RT-PCR analysis. It is recommended that NP be collected twice from both sides of the nostrils or that OP and NP be combined in one VTM to maximize test sensitivity and limit the use of test resources (Center for Disease Control and Prevention, 2020b). The second factor is that the specimen was collected late or very early in the infection.
Hence, it is very important to take specimens at the right time. The third is that the specimen was not properly handled and shipped. Another factor is the technical reasons inherent in the test, e.g. virus mutation or inhibition of PCR (World Health Organization, 2020b).

SPECIMEN SAMPLES
In In the RT-PCR process, there are negative and positive controls. In the negative control, the amplification of viral genes must not be found while in the positive control there must be an amplification of viral genes that are marked by curves. If the RT-PCR negative control is found to be positive, then the PCR process must be repeated (Bustin & Nolan, 2004). The final stage is the reporting, there was indeed a special channel that needed to be done first to report or confirm the results. After interpretation of the results by RT-PCR experts, these results were sent to doctors who will carry out the next stage of verification and validation to report to the hospital and the health service (Espy et al., 2006).

PCR TEST
The false-positive result of the RT-PCR test means that the test is positive even if no virus is present in the sample tested. False-positive results of the RT PCR diagnostic tests are very unlikely. Positive results depend on the sequences of the primers and probes used, and possible cross-reactions with non-target sequences have usually already been ruled out during the design phase (Bustin & Nolan, 2020).
For example, the SARS-CoV-2 rRT-PCR Diagnostic Panel primer and probe sequences were tested for homology with human genome sequences, other coronaviruses, and human microflora to prevent potential false-positive results (Center for Disease Control and Prevention, 2020a). No homology was found with the human genome and microflora sequences, but one of the primers or probes had homology with SARS-CoV and Bat SARS-like coronavirus genome. Since homologies with these coronavirus genomes were not present for the whole set of primers and the RT-PCR reaction probe, a false positive outcome would be unlikely .

PCR TEST
False-negative results from RT-PCR are much more common compared to false-positive results and have more serious consequences in the epidemiological management of the disease (Kalifarhood et al., 2020).
Every RT PCR kits have a limit of detection, ranging from 200-1000 copies/mL, and this is the limit of detection under controlled laboratory conditions. Under real-life conditions, the limit of detection could become higher. Therefore, a low viral load could cause false-negative results. Mistakes during specimen collection, storage, and transportation could result in false-negative results (Jagodzinski et al., 2020).
Variations in the target sequence could cause falsenegative results. The SARS-CoV-2 has evolved into three central variants (Forster et al., 2020). Even though the design of primers and probes has taken into account  (Zou et al., 2020). Negative test results that later became positive were also observed in other longitudinal studies. According to Kucirka et al. (2020), from samples collected from the upper respiratory tracts, no virus could be detected with RT-PCR on the day of infection. On the day of symptom onset, the median false-negative rate was 38%, and the lowest falsenegative rate of 20% was obtained from samples collected three days after symptom onset. Then the falsenegative rate would increase again, reaching 66% at 16 days after symptoms onset.

CONCLUSION
COVID-19 is caused by SARS-CoV-2, an enveloped RNA virus with a large genome (29.9 kb) closely related to human SARS-CoV and bat SARS-CoV. The S protein on the surface of SARS-CoV-2 binds to the ACE2 receptor of the host and promotes entry of the virus into the cell target. ACE2 receptors are present in cells of the arteries, veins, smooth muscles, small intestine, alveoli of the lungs, hair follicles, cardiac myofibroblasts, skin, brain, and kidney, thus SARS-CoV-2 could potentially infect these tissues. The gold standard for detection or SARS-CoV-2 is by rRT-PCR and kits for SARS-CoV-2 detection are available commercially. Proper sampling site, sampling method, the timing of sampling, treatment, and handling of specimens during shipping and storage are all important in obtaining correct test results. Ongoing research is needed to select a better sampling site, timing, and treatment of samples to minimize false results which have serious consequences for the management of the disease.

ACKNOWLEDGMENT
We thank the Universitas Muhammadiyah Bandung for providing support for the completion of this article.