RT PCR test to detect SARS-CoV-2 : 10 major scientific flaws

Source: https://cormandrostenreview.com/report/

Review report Corman-Drosten et al. Eurosurveillance 2020

This extensive review report has been officially submitted to Eurosurveillance editorial board on 27th November 2020 via their submission-portal, enclosed to this review report is a retraction request letter, signed by all the main & co-authors. First and last listed names are the first and second main authors. All names in between are co-authors. External peer review of the RTPCR test to detect SARS-CoV-2 reveals 10 major scientific flaws at the molecular and methodological level: consequences for false positive results. Pieter Borger(1), Bobby Rajesh Malhotra(2) , Michael Yeadon(3) , Clare Craig(4), Kevin McKernan(5) , Klaus Steger(6) , Paul McSheehy(7) , Lidiya Angelova(8), Fabio Franchi(9), Thomas Binder(10), Henrik Ullrich(11) , Makoto Ohashi(12), Stefano Scoglio(13), Marjolein Doesburg-van Kleffens(14), Dorothea Gilbert(15), Rainer Klement(16), Ruth Schruefer(17), Berber W. Pieksma(18), Jan Bonte(19), Bruno H. Dalle Carbonare(20), Kevin P. Corbett(21), Ulrike Kämmerer(22) ABSTRACT In the publication entitled “Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR” (Eurosurveillance 25(8) 2020) the authors present a diagnostic workflow and RT-qPCR protocol for detection and diagnostics of 2019-nCoV (now known as SARS-CoV-2), which they claim to be validated, as well as being a robust diagnostic methodology for use in public-health laboratory settings.  In light of all the consequences resulting from this very publication for societies worldwide, a group of independent researchers performed a point-by-point review of the aforesaid publication in which 1) all components of the presented test design were cross checked, 2) the RT-qPCR protocol-recommendations were assessed w.r.t. good laboratory practice, and 3) parameters examined against relevant scientific literature covering the field.  The published RT-qPCR protocol for detection and diagnostics of 2019-nCoV and the manuscript suffer from numerous technical and scientific errors, including insufficient primer design, a problematic and insufficient RT-qPCR protocol, and the absence of an accurate test validation. Neither the presented test nor the manuscript itself fulfils the requirements for an acceptable scientific publication. Further, serious conflicts of interest of the authors are not mentioned. Finally, the very short timescale between submission and acceptance of the publication (24 hours) signifies that a systematic peer review process was either not performed here, or of problematic poor quality.  We provide compelling evidence of several scientific inadequacies, errors and flaws. Considering the scientific and methodological blemishes presented here, we are confident that the editorial board of Eurosurveillance has no other choice but to retract the publication. CONCISE REVIEW REPORT This paper will show numerous serious flaws in the Corman-Drosten paper, the significance of which has led to worldwide misdiagnosis of infections attributed to SARS-CoV-2 and associated with the disease COVID-19. We are confronted with stringent lockdowns which have destroyed many people’s lives and livelihoods, limited access to education and these imposed restrictions by governments around the world are a direct attack on people’s basic rights and their personal freedoms, resulting in collateral damage for entire economies on a global scale. There are ten fatal problems with the Corman-Drosten paper which we will outline and explain in greater detail in the following sections. The first and major issue is that the novel Coronavirus SARS-CoV-2 (in the publication named 2019-nCoV and in February 2020 named SARS-CoV-2 by an international consortium of virus experts) is based on in silico (theoretical) sequences, supplied by a laboratory in China [1], because at the time neither control material of infectious (“live”) or inactivated SARS-CoV-2 nor isolated genomic RNA of the virus was available to the authors. To date no validation has been performed by the authorship based on isolated SARS-CoV-2 viruses or full length RNA thereof. According to Corman et al.: “We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available.” [1] The focus here should be placed upon the two stated aims: a) development and b) deployment of a diagnostic test for use in public health laboratory settings. These aims are not achievable without having any actual virus material available (e.g. for determining the infectious viral load). In any case, only a protocol with maximal accuracy can be the mandatory and primary goal in any scenario-outcome of this magnitude. Critical viral load determination is mandatory information, and it is in Christian Drosten’s group responsibility to perform these experiments and provide the crucial data. Nevertheless these in silico sequences were used to develop a RT-PCR test methodology to identify the aforesaid virus. This model was based on the assumption that the novel virus is very similar to SARS-CoV from 2003 as both are beta-coronaviruses. The PCR test was therefore designed using the genomic sequence of SARS-CoV as a control material for the Sarbeco component; we know this from our personal email-communication with [2] one of the co-authors of the Corman-Drosten paper. This method to model SARS-CoV-2 was described in the Corman-Drosten paper as follows: the establishment and validation of a diagnostic workflow for 2019-nCoV screening and specific confirmation, designed in absence of available virus isolates or original patient specimens. Design and validation were enabled by the close genetic relatedness to the 2003 SARS-CoV, and aided by the use of synthetic nucleic acid technology.” The Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is an important biomolecular technology to rapidly detect rare RNA fragments, which are known in advance. In the first step, RNA molecules present in the sample are reverse transcribed to yield cDNA. The cDNA is then amplified in the polymerase chain reaction using a specific primer pair and a thermostable DNA polymerase enzyme. The technology is highly sensitive and its detection limit is theoretically 1 molecule of cDNA. The specificity of the PCR is highly influenced by biomolecular design errors. What is important when designing an RT-PCR Test and the quantitative RT-qPCR test described in the Corman-Drosten publication? 1. The primers and probes: a) the concentration of primers and probes must be of optimal range (100-200 nM) b) must be specific to the target-gene you want to amplify c) must have an optimal percentage of GC content relative to the total nitrogenous bases (minimum 40%, maximum 60%) d) for virus diagnostics at least 3 primer pairs must detect 3 viral genes (preferably as far apart as possible in the viral genome) 2. The temperature at which all reactions take place: a) DNA melting temperature (>92°) b) DNA amplification temperature (TaqPol specific) c) Tm; the annealing temperature (the temperature at which the primers and probes reach the target binding/detachment, not to exceed 2 ̊C per primer pair). Tm heavily depends on GC content of the primers 3. The number of amplification cycles (less than 35; preferably 25-30 cycles); In case of virus detection, >35 cycles only detects signals which do not correlate with infectious virus as determined by isolation in cell culture [reviewed in 2]; if someone is tested by PCR as positive when a threshold of 35 cycles or higher is used (as is the case in most laboratories in Europe & the US), the probability that said person is actually infected is less than 3%, the probability that said result is a false positive is 97% [reviewed in 3] 4. Molecular biological validations; amplified PCR products must be validated either by running the products in a gel with a DNA ruler, or by direct DNA sequencing 5. Positive and negative controls should be specified to confirm/refute specific virus detection 6. There should be a Standard Operational Procedure (SOP) available SOP unequivocally specifies the above parameters, so that all laboratories are able to set up the exact same test conditions. To have a validated universal SOP is essential, because it enables the comparison of data within and between countries. MINOR CONCERNS WITH THE CORMAN-DROSTEN PAPER 1. In Table 1 of the Corman-Drosten paper, different abbreviations are stated – “nM” is specified, “nm” isn’t. Further in regards to correct nomenclature, nm means “nanometer” therefore nm should read nM here. 2. It is the general consensus to write genetic sequences always in the 5’-3’ direction, including the reverse primers. It is highly unusual to do alignment with reverse complementary writing of the primer sequence as the authors did in figure 2 of the Corman-Drosten paper. Here, in addition, a wobble base is marked as “y” without description of the bases the Y stands for. 3. Two misleading pitfalls in the Corman-Drosten paper are that their Table 1 does not include Tm-values (annealing-temperature values), neither does it show GC-values (number of G and C in the sequences as %-value of total bases). MAJOR CONCERNS WITH THE CORMAN-DROSTEN PAPER A) BACKGROUND The authors introduce the background for their scientific work as: “The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the outbreak is more widespread than initially thought, and international spread through travelers does already occur”. According to BBC News [4] and Google Statistics [5] there were 6 deaths world-wide on January 21st 2020 – the day when the manuscript was submitted. Why did the authors assume a challenge for public health laboratories while there was no substantial evidence at that time to indicate that the outbreak was more widespread than initially thought? As an aim the authors declared to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available. Further, they acknowledge that “The present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.” B) METHODS AND RESULTS 1. Primer & Probe Design 1a) Erroneous primer concentrations Reliable and accurate PCR-test protocols are normally designed using between 100 nM and 200 nM per primer [7]. In the Corman-Drosten paper, we observe unusually high and varying primer concentrations for several primers (table 1). For the RdRp_SARSr-F and RdRp_SARSr-R primer pairs, 600 nM and 800 nM are described, respectively. Similarly, for the N_Sarbeco_F and N_Sarbeco_R primer set, they advise 600 nM and 800 nM, respectively [1]. It should be clear that these concentrations are far too high to be optimal for specific amplifications of target genes. There exists no specified reason to use these extremely high concentrations of primers in this protocol. Rather, these concentrations lead to increased unspecific binding and PCR product amplification. Table1: Primers and probes (adapted from Corman-Drosten paper; erroneous primer concentrations are highlighted) 1b) Unspecified (“Wobbly”) primer and probe sequences To obtai