Why Real Time PCR gives false negative results for clinically confirmed corona patients?
Zahir Abbas Hilmi
*Department of Biocheistry and Molecular Biology, Faculty of Science, Gezira University
* Medicine Program, Napata College
In November, 2002, an epidemic caused by a novel Betacoronavirus – SARS –CoV 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-CoV . 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- CoVID 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-CoVZC45 (29732bp) and bat-SL-CoVZXC21 . 2019-nCoV is less genetically similar to SARS- CoV (79%); with a genome of 29751bp. 2019-CoV The genome of MERS-CoV (30,119 bp) was found to be the least related 50% to 2019- CoV.
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-CoVZC45 and bat-SL-CoVZXC21, 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
1- it’s operation requires highly skilled experts to avoid any mistakes
2- The site from which the sample taken not from the right place or no viruses in it
3- 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.
4- 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 )
5- 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.
6- 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
7- 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)