Outbreak of pneumonia in the third week of December-2019, in Wuhan City of Hubei Province of China led to the isolation of a new type of coronavirus in January 2020. The same was named as novel coronavirus-2019 (2019-nCoV) by Chinese Health Authorities. Pathogen free 'Human Airway Epithelial (HAE) cells' in tissue culture bottles were apically inoculated with bronchoalveolar lavage fluids and/or throat swabs from 9 patients. Apical supernatants from these cultures were the source of viral RNA. Genomic sequencing of 2019-nCoV was done by Chinese Scientists using ‘Sanger Sequencing’ facility and made available to GenBank and is registered under GenBank accession ID: MN908947. In 5 cases out of 9 Illumina and Oxford Nanopore Sequencing was also done. All the 9 patients whose samples were used to isolate virus for genomic sequencing had exposure to Huanan Seafood Market in Wuhan City. One of these patients stayed near Huanan Seafood Market.
Genomic implications:
There exists homology in genetic sequences of six strains on 2019-nCoV. The GenBank accession IDs of these strains are: EPI_ISL_402019, EPI_ISL_402020, EPI_ISL_402021, EPI_ISL_402022, EPI_ISL_402023 and EPI_ISL_402024. These epidemic strains of COVID-19 virus are just like human siblings.
It has been documented that Genomic sequences of 2019-nCoV obtained from nine patients of severe pneumonia at Wuhan City of Hubei Province of China had 88% identity with two bat-derived 'Severe Acute Respiratory Syndrome’ (SARS) like coronaviruses with genomic IDs: bat-SL-CoVZC45 and bat-SL-CoVZXC21. Genetic sequence of 2019-nCoV has been found very distinct from SARS-CoV (2002-03). Findings were published by Chinese group of scientists in the Lancet, volume 395 in the month of February 2020.
A group of veterinary scientists working
under the guidance of Yongyi Shen of the College
of Veterinary Medicine, South China Agricultural University, Guangzhou-510642,
China, have explored through extensive study on 25 Malayan pangolins (Manis
javanica) and 4 Chinese pangolins (Manis pantadactyla) that 2019-nCoV
has very close relationship with coronavirus isolated from 17 out of 25 Malayan
pangolins. However, Chinese pangolins were found negative for 2019-nCoV
infection by RT-PCR. Coronavirus isolated from Malayan pangolins showed 100%,
98.2%, 96.7% and 90.4% amino acid identity with 2019-nCoV in E, M, N
and S genes, respectively. The study reflects that Malayan
pangolins are the natural host of this zoonotic coronavirus. Yongyi Shen
and his team has proved through this marvelous study that 2019-nCoV is
an identical twin of Pangolin-CoV.
Analysis of plasma samples from 8 of the Malayan
pangolins showed high positivity for antibodies against 2019-nCoV by double
antigen sandwich ELISA. Plasma sample of one of the Malayan pangolins showed
positivity for antibodies against 2019-nCoV even at 1:80 dilution of plasma.
The Real Time-Reverse Transcription-Polymerase Chain Reaction (rRT-PCR) was developed for early detection of 2019-nCoV infection using nasopharyngeal and/or oropharyngeal swabs from patients having clinical symptoms of viral infection. RT-PCR has limitations that it could not be used for detecting past infections.
Diagnostic dilemma is that rRT-PCR covers all the six strains of 2019-nCoV. There could be more unknown strains of 2019-nCoV having identical genomic expression. Molecular diagnostic technology is very new and sensitive but has its limitations. It provides indirect evidence of a causative organism. Real Time RT-PCR for 2019-nCoV does not distinguish between active infection or positive results due to unknown SARS Related Coronaviruses (SARSr-CoV).
The only validated method for diagnosis of 2019-nCoV by World Health Organization (WHO) is rRT-PCR and nobody could question its accuracy. Collection of sample, extraction and purification of viral RNA, storage of reagents of RT-PCR Kits, setting up of rRT-PCR Assay, operation of PCR-Machine and interpretation of rRT-PCR results need extensive training and experience.
Information Brochures/Manuals of all the Real Time RT-PCR Kits used for detecting 2019-nCoV immaculately exhibit that the kits are meant for Research use only (RUO) . Information is available online regarding recommended use of kits. I would like to share an adapted image from the Brochure of RealStar SARS CoV-2 RT-PCR Kit 1.0 of Altona Diagnostics GmbH, Hamburg (Figure-1) and another image from the Manual of Liferiver Novel Coronavirus (2019-nCoV) Real Time RT-PCR Kit of Shanghai ZJ Bio-Tech Co. Ltd., China (Figure-2), for attesting my point that diagnostic use of RT-PCR kits for 2019-nCoV (SARS CoV-2) is not valid or ethical.
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| Figure-1 For Research use only (RUO) |
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| Figure-2: For Research use only (RUO) |
Extraction of RNA is the start point of all the kits for RT-PCR analysis of nasopharyngeal or oropharyngeal swabs as well as bronchoalveolar lavage samples. The purity of RNA extracted could only ensure the accuracy of results. It is highly important to use the RNA Extraction Kit compatible with the kit being used for rRT-PCR. Some most extensively used RNA Extraction Kits and Systems are:
§ AltoStar Automation System AM 16 (Altona
Diagnostics)
§ QIAamp Viral RNA Mini kit (QIAGEN)
§ QIAsymphony (QIAGEN)
§ MagNA Pure 96 System (Roche)
§ Maxwell 16 IVD Instrument (Promega)
§ VERSANT kPRC Molecular System SP (Siemens
Healthcare)
Real Time RT-PCR:
The rRT-PCR is a complex assay for detection of target genes: Target-E Gene (for B βCoV specific RNA), Target-S Gene (for SARS CoV-2 specific RNA) and Target-IC (for Internal Control). Different companies have different reporters (fluorescent dye labels) with or without quencher. The RealStar SARS CoV-2 RT-PCR Kit has used FAMTM, Cy5 and JOETM without any quencher respectively for above mentioned target genes.
The RT-PCR comprises three steps, which are completed in Thermal Cycler at different temperatures ranging from 55oC to 95oC. The dye acquisition takes place during amplification cycle. In brief:
Step-I is Reverse Transcription; it is done at 55oC at hold for single cycle of 20 minutes.
Step-II is Denaturation; it is done at 95oC at hold for single cycle of 2 minutes.
Step-III is Amplification of DNA; it is a multicycle process (at least 30-45 cycles). One cycle of Amplification is completed in holds at three different temperatures: 95oC for 15 seconds, 55oC for 45 seconds and 72OC for 15 seconds. So, one amplification cycle is completed in 1 minute and 15 seconds. 45 cycles for amplification will be completed in 56 minutes and 25 seconds (75x45=3375 seconds). So, the total turnaround time for rRT-PCR is 3 hours including sample preparation. However, it would take longer as the kit is meant for 14 samples and 6 controls. Preparation of multiple specimens and setting up machines needs extra time and precautions to avoid contaminations.
Some most commonly used RT-PCR machines are:
- ABI Prism 7500 SDS (Applied Biosystems)
- CFX96TM Deep Well Real-Time PCR Detection System (Bio-Rad)
- CFX96TM Deep Well Dx System (Bio-Rad)
- CFX96TM Real-Time PCR Detection System (Bio-Rad)
- CFX96TM Dx System (Bio-Rad)
- LightCycler 480 Instrument II (Roche)
- Rotor-Gene 6000 (Corbett Research)
- Rotor-Gene Q5/6 Plex Platform (QIAGEN)
- Mx 3005TM QPCR System (Stratagene)
- VERSANT kPCR Molecular System AD (Siemems Healthcare)
The data analysis on Real Time-PCR machines needs expertise
as per instrument manufacturer’s instructions. Qualitative Analysis of fluorescence
signals captured for genes under detection is done to conclude the outcome of rRT-PCR
assay.
Past infection with 2019-nCoV could be detected trough tests for detecting IgM and IgG type antibodies against the virus. Double antigen sandwich ELISA could be very sensitive and specific assay for detecting both the IgM and IgG type antibodies.
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