Analysis of tests used in detection/diagnosis of SARS-CoV-2/COVID-19

The detection molecular biology that The Polymerase Chain detection and of speciﬁc DNA or RNA (through cDNA) molecules . The technique is based on ampliﬁcation of regions that are determined by using speciﬁc primers (oligonucleotides). the DNA/cDNA sequence present, the bind that, and then enable the ampliﬁcation of the while evolution Real-Time include also nucleic acid isothermal ampliﬁcation and imaging methods (X-ray). Although scientists can use diﬀerent methods for detection, they should always take into consideration potential limitations of them. This review describes the widely used diagnostic tests for COVID-19 detection and their advantages and weaknesses. The Centers for Disease Control and Prevention (CDC) suggest the use of molecular biology techniques for detection of SARS-CoV-2. The test is based on Real-Time Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR), and consumables are available from diﬀerence companies (BioSearch Technologies, KIT-nCoV-PP1- 1000, Integrated DNA Technologies (IDT), 10006606,). The basic principle behind that test is the detection of the virus nucleocapsid gene (N), using speciﬁc primers and probes. The test includes another set of primers/probes for the detection of the human RNase P gene (RP) as internal control. The sampling is performed with isolation of specimens from upper and lower respiratory systems (nasopharyngeal or Latest weeks the humanity is faced with the spread of a new coronavirus, SARS-CoV-2 that causes a respiratory illness with high mortality rates, COVID-19. Since there is no approved treatment or vaccination against that speciﬁc coronavirus the reduce in virus spread is essential. That is based in the use of appropriate tools, enabling the accurate and early detection. Molecular biology and immunological techniques are widely used in order to predict the COVID-19 cases in a very short period of time. These are commonly based either in identiﬁcation of the SARS-CoV-2’s genetic material or in detection of antibodies that have been produced by the immune system against the virus. Many of the above mentioned tests have been validated and approved by local authorities. However, there are much more companies that provide detection tests, without basic validation processes, contributing in non-precise data. The present review aim to analyze the most common platforms that are used in COVID-19 detection, analyzing their advantages and weaknesses. Therefore, each physician will be equipped with appropriate information required for each test. The use of Real-Time RT-PCR is very sensitive and able to identify the genetic material even at very low amounts. However the main limitation is the requirement of specimens only from upper and lower respiratory system. It is also suggested to collect specimens (types and time points) from the same patient, since a negative results should checked again, and does not quarantine absence of infection. The test has not been established from blood specimens while there is a limit of detection at 10-5 copies/ul or RNA. On the other hand, the use of three diﬀerent primer/probe sets increase the accuracy or results, compared with other tests targeting only one region. The above diagnostic kit has been tested in other viruses and pathogens and ensuring no signiﬁcant combined homologies with them or any other part of human genome.


RNAse P Forward
Primer AGA TTT GGA CCT GCG AGC G [7] RNAse P Reverse Primer GAG CGG CTG TCT CCA CAA GT FAM -TTC TGA CCT GAA GGC TCT GCG CG -BHQ-1 The use of Real-Time RT-PCR is very sensitive and able to identify the genetic material even at very low amounts. However the main limitation is the requirement of specimens only from upper and lower respiratory system. It is also suggested to collect specimens (types and time points) from the same patient, since a negative results should checked again, and does not quarantine absence of infection. The test has not been established from blood specimens while there is a limit of detection at 10-5 copies/ul or RNA. On the other hand, the use of three different primer/probe sets increase the accuracy or results, compared with other tests targeting only one region. The above diagnostic kit has been tested in other viruses and pathogens and ensuring no significant combined homologies with them or any other part of human genome.

RNAse P Probe
Up to now there are more than 250 available tests based on PCR reactions, but only 98 are CE-IVD (In vitro diagnostic). Due to the circumstances many of the kits are authorized as Emergency Use Authorization should be followed also in all diagnostic kits .
Another important issue for all kits is the lack of clinical evidence, concerning positive and negative predictive values (PPV-NPV). PPV is defined as the probability of a test to be positive, when the sample has the specified disease, while NPV referred to the probability of a negative test, when it is really negative .
The calculation of PPV and NPV requires the blind test of the kit in patients and healthy volunteers. There have been recorded cases where kits have withdrawn or revised since the sensitivity of the assay has been found to be lower than expected.
The National Authorities have published guidelines not only for the diagnostic tests, but also for the biosafety of laboratories. In general, all PCR laboratories should be divided in pre and post-PCR areas reducing the chance of contamination. On the pre-PCR area is taking place the nucleic acid extraction and preparation of reaction, while the post-PCR is appropriate for amplification and post-amplification processes . The processing of suspect specimens should be performed on a BSL-2 laboratory (Biosafety level-2), where virus isolation in cell culture must be conducted in BSL-3 laboratory . Inappropriate sampling conditions put in jeopardy both healthcare stuff and healthy individual as well.
Specimen type is one major issue regarding molecular diagnostic tests. Since SARS-CoV-2 infects mainly upper and lower respiratory system it is suggested sampling of upper and lower respiratory tract.
Therefore it is recommended using nasopharyngeal or oropharyngeal swabs, nasal aspirate specimen, nasal mid-turbinate swab or anterior nares specimen. Regarding the lower respiratory tract it is recommended bronchoalveolar lavage, tracheal aspirate, pleural fluid, lung biopsy or sputum. Based on

Biosafety of Laboratories
[11] [12] Sampling recent data the viral load is detected soon after symptoms onset and the load is higher in nose than in throat . In addition the use of lingual swabs, although is easier to use and requires no-expertise personnel, provide lower positive rate than throat swabs. On the same study it was demonstrated that training or experience of personnel might affect the sampling process . In general the higher positive rates have been shown in bronchoalveolar lavage fluid specimens (93%) followed by sputum (72%) and nasal swabs (63%). The positive rates decrease in other types of specimens as follow: fibrobronchoscope brush biopsy (46%, pharyngeal swabs (32%), feces (29%), blood (1%) and no detection in urine .
Therefore the use of different specimen types for each individual is highly recommended. It is noteworthy that has been detected a patient with meningitis/encephalitis, where SARS-CoV-2 was not detected in nasopharyngeal swab but was detected in a cerebrospinal fluid (CSF) . Although that is surprisingly, SARS coronavirus RNA has been detected in the CSF of a patient with severe acute respiratory syndrome a few years ago .
Another method used for detection of SARS-CoV-2's RNA is the isothermal nucleic amplification, which is not limited by the constraint of thermal cycling. It has been used in the past for rapid detection of severe acute respiratory syndrome coronavirus , however only a few data are available about specificity and sensitivity against SARS-CoV-2 detection.
Detection of SARS-CoV-2 is not limited in RNA detection and identification but also in serology tests, based on detection of specific antibodies produced by the immune system. Immunoglobulins (  Isothermal nucleic amplification [18] Serology Tests [19] [20] [21] [22] [23] Conclusion is of primary importance, since inappropriate specimens lead to negative results. The combination of specimens from different parts of upper or lower respiratory tract increase the specificity of assays. It is also noteworthy that all procedures should be conducted on laboratories under restrict biosafety levels, ensuring protection of personnel and contamination of tested samples. Scientists and healthcare experts providing molecular diagnostic tests need to obey the rules of MIQES, including appropriate controls in each case. On the contrary, diagnosis of COVID-19 based on antibodies detection, is very simple, requiring blood, plasma or serum and does not implicate specialized equipment. Nevertheless, the detection of antibodies supposes activation of the immune system, therefore in most of the times symptoms.
Serological tests are more suitable diagnosis of individuals with symptoms, rather than for screening.
Recapitulating this brief review, each test has both advantages and disadvantages that must be taken into from national authorities and healthcare experts. Reliable and accurate tests are preferable to simple and quick tests.