**8. Future prospects of RT-PCR**

PCR method was discovered in 1986 and since then the method is serving medical sectors. In the future, as potential molecular diagnostic methods, PCR will play a significant role. Lots of PCR methods are already used in various research and medical fields, but as we know currently under this pandemic situation RT-PCR has turned out to be a boon in healthcare sectors. Lots of kits have been manufactured throughout the world with some or little variation, thus making it more sensitive, specific and less time consuming. One factor which essential under pandemic situation was a multiple sample analysis in one go [5]. Thus more focus on using portable POC systems, we can imagine the use of micro-fluidics doing concurrent multiple sample analysis. It is possible to analyse immunomagnetic exosomal RNA by using micro-fluidic systems RT-PCR said to be a Chip-based integrated real-time reverse transcription PCR platform [70]. Similar way chip-based RT-PCR digitally can quantify the mRNA in single cell [71].

Scalable, quick, and inexpensive diagnostics of COVID-19 by RT-PCR could help restrict the spread of SARS-CoV-2, saving lives as a result. RNA extraction, however, constitutes an obstacle to the scale-up of experiments. Thus, one research was done directly using the RT-PCR and heat inactivated sample and efficacy was tested. The study proved that is not necessary to carry RNA extraction testing. The study also suggests the use of standard protocols for RT-PCR and transport media by the whole world so the it will become easier to deal with future epidemics. Such RT-PCR said to be an hid RT-PCR (heat-inactivated direct RT-PCR). Sample collection and rather than using transport media, usage of lysis buffer is found to be more efficient as it will directly lys the sample and can be used in RT-PCR without a need of any RNA extraction kit [72].

As *Coronaviridae* family consist many RNA viruses which not only infect the humans but also animals, birds etc. [73]. If we look at the history of this family will see that it has been always serious when it started infecting like in 2002, Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged, in 2012, Middle East respiratory syndrome coronavirus (MERS-CoV), in 2016, swine acute diarrhoea syndrome coronavirus (SADS-CoV) and the latest one which cause pandemic in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [74]. Among this SADS-CoV infects pigs while other infect humans [75]. This all was controlled well but current 2019 epidemic was beyond control due to lack of diagnosis, medicines and vaccine. For now after all efforts diagnosis for SARS-CoV-2 has been found but as lots of virus under this family can emerge in future and which has already spread in past needed to be studied [76]. So one research was completely done to make a molecular diagnostic kit which can identify almost all CoVs, thus to diagnose easily any virus among this family in future [77]. They designed a semi-nested RT-PCR based upon 38 genome sequences that has been recorded from human and animal CoVs. Thus proved to be a great finding which can diagnose all available CoVs or which can emerge in future too [77].

Lots of research regarding RT-PCR has been done, PCR like nested PCR, ddPCR, two step RT-PCR is an advance and gives more accurate results but labs were not that equipped and it was not possible to set up everything under such pandemic situation. Hence if we think about future this all equipments and facilities have to be adopted by laboratory to deal with emerging epidemics [57].

#### **9. Conclusion**

SARs-CoV-2, originated from Wuhan, China and spread all over world, causing a pandemic situation which affected the whole world badly at economic, social,

medical level also. Initially it was very difficult to deal with virus as no diagnosis, treatment or vaccine was available, but after lots of efforts of researchers now we have a good diagnosis and control condition in regards spread of infection. Even vaccination has been started in almost all countries. As in the earlier period of pandemic, the diagnosis was the main factor to prevent the spread of the virus. So main focus was on a diagnostic that to on molecular diagnosis as it is more efficient and accurate way of detection. Currently use molecular method is RT-PCR also said to be a gold standard detection method. Lots of RT-PCR kits are now available in most of the countries with little modifications and approvals. The key point to be noticed before the laboratory experiments is the right reliable sampling. Nasopharyngeal and oropharyngeal swabs, which are safer for collection are recommended for screening or early detection. If we talk about a future of RT-PCR advancement in methods like nested PCR, ddPCR, two step RT-PCR have been already done, which is found to be more accurate, but lack of instrumentation and expertise have put it behind, but in future this thing has to be focused and implemented so that the world can deal with any future epidemic.
