Complex to simple technologies in Molecular Diagnosis of COVID-19
Molecular-based In-Vitro Diagnostics (IVD) testing came to limelight due to COVID-19 as it is the only technology that accurately detects the coronavirus and aids in containing the pandemic. If the pandemic is not contained it will spread to a huge number of individuals and increase the chance of evolution of the virus. This may lead to virulent variants leading to serious epidemics that happened in the past.
Importance of Molecular Diagnostics (MDx)
The principle of Molecular Diagnostics (MDx) test was based on nucleic acid amplification where the target gene of importance is amplified from single copies to million copies and detected with an optical unit. At present two major technologies are used for the nucleic acid amplification; Polymerase Chain reaction (PCR) and Isothermal reaction-based amplification or Loop Mediated isothermal Amplification (LAMP). Since COVID-19 is caused by the RNA virus, SARS-CoV-2 a reverse transcription step is mandated before PCR or LAMP, thus called on polymerase chain reaction (RT-PCR) RT-PCR or RT-LAMP.
The first step in any of the nucleic acid amplification tests is the extraction of nucleic acid (RNA/DNA) from the sample. The principle is to extract, purify and to concentrate the nucleic acid in the sample to have faster and accurate amplification of the target gene without any loss of the target. Gold standard confirmatory MDx assays need extraction of DNA and RNA which is unavoidable.
Nucleic Acid Extraction, Purification and Concentration
There are many methods for the nucleic acid isolation, like centrifugation-based extraction using columns, it’s time consuming. But quick detection of the targets is very important. To increase the speed of nucleic acid extraction instead of the centrifugation method, magnetic beads are used to capture, purify and concentrate the nucleic acid which is done in 10 to 30 minutes unlike the centrifugation method which needs at least 60 minutes. Magnetic bead-based methods are very fast, simple and easily automated in simple nucleic acid extraction instruments like MISPA Magenta manufactured and marketed by Agappe Diagnostics Ltd, Kerala, India.
Today, the most common method used for nucleic acid extraction in almost all COVID-19 testing laboratories are magnetic bead-based methods. This principle is based on the property of nucleic acid to bind to magnetic beads based on the charge. The magnetic beads with nucleic acid are then isolated using a magnetic road washed and eluted in nuclease free water to release the nucleic acids. Moreover, to make the process simple, prefilled cartridges are available in the magnetic bead method to speed up the process like ACM Auto, (Prefilled cartridges form Agappe Diagnostics Ltd, Kerala India). Only sample addition steps are done manually. The technology is so evolved in such a way that the extractions are completed within 10 minutes using ACM Auto – Prefilled cartridge in MISPA Magenta or in any magnetic bead-based nucleic acid extraction system.
Amplification of SARS CoV-2 Viral Target
The accuracy of RT-LAMP/RT-PCR depends on the quality of RNA extracted from the given swab samples. Laboratories should make sure that the extraction process is performed perfectly. It is always advisable to use an extraction kit with more than 95% recovery rate and having an extraction limit of 1 copy/uL which is very crucial for the proper amplification, like Agappe Diagnostics Ltd Extraction kits in MISPA Magenta extraction system. In most of the cases, missing the viral target amplification is mainly due to the low recovery during the extraction. It is recommended to repeat the extraction, in case of inconclusive or abnormal results.
Earlier, the nucleic acid amplification was done using RT-PCR technology, where the amplification was made with the help of thermal cyclers. Thermal
cyclers are capable of cycling temperatures as required. The early thermal cyclers were water baths with three different temperatures and the reaction sample was moved periodically. Detection of the amplification was done separately which required high technical expertise and time.
Nucleic Acid Amplification Technologies
Technological Innovations has made compact systems in molecular segment where the RT-PCRbased instruments become very compact with possibilities of performing 1 to 1536 samples at a time which made MDx segment gaining newer heights but still some factors like huge cost and complex principles limited the access of such technologies only to the premium laboratories till the beginning of COVID-19.
Loop Mediated isothermal Amplification (LAMP) technique was introduced in the year 2000 in Japan. This technology was intended for quick target amplification using single (iso) temperature. Since there is no need for complex thermal cycling hardware, the LAMP-based systems are highly compact, cost effective and hence got wide acceptance around the world. Because of the simplicity, affordability and accuracy the LAMPbased systems can be used for faster testing for the masses during the present pandemic. Agappe Diagnostics Ltd India has launched RT-LAMP COVID detection kit – LUME Screen nCoV and MISPA LUME a Real time LAMP analyzer for the first time in India supporting the Indian government to fight the pandemic.
One of the advantages of LAMP-based assay over the conventional PCR is the time taken for amplification. In case of COVID-19, the LAMPbased
assay amplifies the samples within 10 minutes after sample addition and the entire process starts generating reports from 10 mins onwards and gets completed within 35 minutes (in conventional standard PCR takes 90 to 120 minutes). Since there is no separate cool down cycle required as in others, we can immediately load the next batch of samples in the LAMP analyzer. The next generation LAMP-based systems are believed to be in microfluidics platform bringing down the size of the instrument considerably.
Apart from the advancements in the hardware, a lot of innovations are made in the amplification reagents. Majority of the PCR-based reagents are thermolabile requiring strict temperature control (-20 0C), it’s one of the major limitations for storage/transport. Reagent manufacturers are trying to make the reagents in lyophilized format and only few have succeeded. LAMP-based assays are available in lyophilized format, which is quite stable between 2 and 8 0C.
Agappe Diagnostics Ltd has come up with a more advanced kit called AG InstaLUME-based on the continuous experiments conducted and from the inputs of various COVID-19 testing Labs. Many laboratories were overwhelmed by the huge sample loads received for processing during the peak of the first and second waves of the pandemic. The huge sample load was above the capacity of many labs leading to huge delay in reporting and containing the pandemic. Hence, Agappe Diagnostics came up with a solution – AG InstaLUME in which RNA extraction is not needed. The swab sample can be collected directly in the lysis buffer. RNA containing buffers can be immediately taken for the RT-LAMP assay in MISPA LUME. From 10 minutes onwards results will be generated by MISPA LUME. This is very appropriate for mass screening of travellers in places like Airports, Railway and Bus stations and in other possible gathering places like Malls, Cinemas, etc. In hospitals, AG InstaLUME can generate reports accurately in 10 to 15 minutes where patients need not wait for any emergency procedures for 24 hours for the RT-PCR report which may increase the complications in patients.
Future Developments
With the entry of microfluidics and other related technical innovations into MDx, new pocket sized, handheld, home-based molecular detection systems will be soon available to all. This future technology will decentralize the MDx tools to all, unlike its availability only in premium laboratories at present.