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Short Communication on Coronavirus 2019-nCoV

  • Events
  • Short Communication on Coronavirus 2019-nCoV

Short Communication on Coronavirus 2019-nCoV

  • 15 Dec 2020
  • Category: Events
  • 447 Viewer

?Why Real Time PCR gives false negative results  for clinically confirmed corona patients

Dr. Zahir Abbas Hilmi

 

Department of Biocheistry and Molecular Biology, Faculty of Science, Gezira University*

Medicine Program, Napata College*


November, 2002,  an epidemic caused by  a novel  Betacoronavirus   – SARS –nCoV emerged in Guangdong, southern China.  SARS or severe acute respiratory syndrome, resulted in   more than 8000 human infections and 774 deaths in 37 countries during 2002–03

In 2012, the  Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), which was first detected in  Saudi Arabia. MERS infected 2494 patients and kills  858 since September, 2012, including 38 deaths following a single introduction into South Korea

In 2019  December,  a new human-infecting betacoronavirus  2019-nCoV  pandemic started in  Wuhan in China. 2019-nCoV  is   sufficiently divergent from SARS-nCoV . The  phylogenetic analysis suggests that bats might be the original host of this virus (Lu et. al., 2020). The bats were likely to be the reservoir for 2019-nCoV  as it is most closely related to other betacoronaviruses of bat origin

The new pandemic 2019- nCoVID  started in December 2019 and  up to  24 March 2020,       within 67 days the number of infected patients in 196 countries was 410,213 , with 18,266 deaths, 107,182 recovered. The new pandemic now is out of control and the numbers of victims increased  in a logarithmic way

The  coronavirus  epidemic in the world started  in Wuhan  in China, caused by a new novel  type of the Family Coronaviride  the  2019  nCoV. According to Baltimore’s nucleic acid based taxonomy of viruses, members of Coronaviridae  belonged to Group IV positive single stranded RNA viruses (+ssRNA). Coronavirus is the largest  RNA viruses their genome  ranged from 26000bp to 32000bp . The Coronaviridae  genome is replicated by RNA dependant RNA polymerase , that induce more mutations 1 in  every 1000 base pairs

The complete  genome sequences    of  the novel virus  2019-nCoV was 29,844 bp and was  compared to genome other coronaviruses ( Lu et. al., 2020).  The genome sequence  of  2019-nCoV  is most closely related (87.237 %) to two  bats coronovirus that collected  2018 in Zhoushan, eastern China  ; Bats- SL-nCoVZC45 (29732bp) and bat-SL-nCoVZXC21  .  2019-nCoV    is less  genetically similar to SARS- nCoV (79%); with a genome of 29751bp.  2019-nCoV   The genome of  MERS-nCoV  (30,119  bp)  was found to be the least  related 50% to  2019- nCoV

Phylogenetic analysis revealed that 2019-nCoV fell within the subgenus Sarbecovirus of the genus Betacoronavirus, with a relatively long branch length to its closest relatives bat-SL-nCoVZC45 and bat-SL-nCoVZXC21,  Accordingly, the realtime PCR kits or  other immunodiagnostic kits might not be able to detect the new virus with high percentages of false negatives due to sequence variation

Diagnosis of  2019 nC0VID is very important to find out the first few cases and to isolate them, to prevent unchecked community spread. Confirmation of clinical diagnosis and  follow up   of viral load  in patients before and after treatment, to ensure complete cure

Many scientific reports showed that early Chinese may have had false negative rate as high as 50%. In USA many test kits released  by the CDC on Feb.2020 were defective. Accordingly, the magnitude of this epidemic is still unknown. Also lower numbers of people were tested

?Why Real Time PCR gives false negative results for patients infected with CoVID 2019

Though real time PCR is the most advance and sensitive molecular diagnostic test

it’s operation requires highly skilled experts to avoid any mistakes *

The site from which the sample taken not  from the right place or no viruses in it *

The samples not preserved  well or transported  in unsuitable preservatives *

that may destroy the virus RNA genome or this preservatives may alter or inhibits PCR enzymes May be sterilization measures affect the sample, The RNA extraction Kits may not be suitable of efficient to obtain  coronavirus RNA nucleic acid in good quality or with high concentration

The test has two successive steps : the first one to do reverse transcriptase real time PCR to produce virus *

cDNA, and the second to use the cDNA virus for amplification

The two steps based on prior knowledge about the conserved sequence of the new coronavirus so as to design the PCR primers

The  rt PCR primers  designed  were not completely complementary to the new coronavirus nCoV ID 2019 , or not designed  from the   conserved sequences of RNA  genome of the new coronavirus ، may be designed for old other corona virus that  not suitable for the detection of the  new coronavirus.(i.e. older RT PCR kits may be used )

The  annealing or hybridization  temperature  for RT.PCR primers is not adjusted (calibrated) may be very *

high

that may give false negative results on clinically positive corona patients

In this situation a new  type specific  primers from the nCoV ID 2019  conserved sequence , should be designed . Moreover,  TagMan probe based real time PCR  primers (type specific) should be used

The Concentration of the virus nucleic acid in step one reverse transcriptase PCR  *

Or step 2 for cDNA may be  very low and below 100 nanogram/microliter

According to Lu et. al., 2020, the following primers should be used. The specific *

primers and probe set (labelled with the reporter 6-carboxyfluorescein [FAM] and the quencher Black Hole Quencher 1 [BHQ1]) or orf1a were as follows

Forward primer 5′-AGAAGATTGGTTAGATGATGATAGT-3′

Reverse primer 5′-TTCCATCTCTAATTGAGGTTGAACC-3′

Probe 5′-FAM-TCCTCACTGCCGTCTTGTTGACCA-BHQ1-3′

Internal control, the human GAPDH gene

Forward primer 5′-TCAAGAAGGTGGTGAAGCAGG-3′

Reverse primer 5′-CAGCGTCAAAGGTGGAGGAGT-3′

Probe 5′-VIC-CCTCAAGGGCATCCTGGGCTACACT-BHQ1-3′



Dr. Zahir Abbas Hilmi

PhD Molecular Virology (Division Taxonomy of Human Papillomavirus, Department of Tumor Virology , the DKFZ German Cancer Research Center & Gezira University)


 

Reference

Lu, R. et. al.,( 2020) . Genomic characterization and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. The Lancet, (395)

DO  – 10.1016/S0140-6736(20)30251-8

  • 15 Dec 2020
  • Category: Events
  • 447 Viewer