Since November 2019, the world has had to face an unprecedented public health emergency: the coronavirus: SARS CoV 2, now COVID-19 [1, 2] .
The pandemic has put a strain on the health system in its entirety and above all has put microbiologists in serious diagnostic difficulty, having to answer questions without having valid scientific evidence, especially in the early stages [3] [4] [5] . Unfortunately to date, even in our country, and with important differences from region to region, the problem persists.
On one hand, we have to deal with the population legitimate request to access the testing and their availabilities; on the other hand we have to face the same issue from health institutions and we do have to put up with the tech the technological and diagnostic resources actually available. All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted June 19, 2020. . https://doi.org/10.1101/2020.06.17.20124396 doi: medRxiv preprint Furthermore, another key element is also the problem of limited human resources, engaged in microbiology laboratories to carry out the COVID19 tests.
After the moment of maximum spread of the virus, the real challenge for Italy as for the rest of the world, steered into the stocking of the diagnostic tests, the simplicity in the administration of the latter to the population and the sustainability of the system as a whole.
In a time of great diagnostic difficulty, our research team decided to design a new diagnostic system that could satisfy the required sensitivity and specificity, but allowing at the same time a reasonable rapid diagnosis, thus it could be introduced in a diagnostic process matching up to the requests of the health system. Our assay (from now kit) is a qualitative Reverse Transcriptase Real Time PCR probe-based (qRT-PCR) detecting target genes of COVID-19. In this paper we present the characteristics of our system. All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted June 19, 2020. A portion of an endogenous gene of human β-actin was used as internal control (IC) for the test, the latter also allowed the evaluation of correct nasopharyngeal sampling.
In order to evaluate our kit an initial proficiency assay was performed at Microbiology laboratories-Department of Experimental Medicine of "Tor Vergata" University of Rome. This proficiencyassay was run using a chimeric plasmids (CPs) in which sequences of the virus were artificially inserted on a plasmid [pBlueScript II SK(+)]. Synthesis of CPs was hired to Bio-Fab Research (Bio-Fab Research, Rome, Italy). All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted June 19, 2020. Ideal condition of reaction was achieved using for 20 μl reaction a master mix composed as follow:
2X qPCRBIO probe 1-step Go Mix 10 ml; ppMix (Mix of all primer and probe) 5 μl; 20X RTase Go 0,2 μl; and 5 μl of specimen. In particular ppMix contained as final concentration: 10 picomoles All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted June 19, 2020. RT-PCR conditions, for both instruments, were the following: one step 45°C for 10 minutes; one step at 95°C for 2 min; 40 steps at 95°C for 5 seconds and the last one at 60°C for 25 seconds.
The instrument was programmed to read gene RdpR on Fam, gene E on Rox, gene N on Cy5 and in β-actin on Hex channel. All rights reserved. No reuse allowed without permission.
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The positivity in our test was based on WHO guidelines [6]. In particular, a sample was considered positive if showing a signal in at least one fluorophore Rox (gene E) and/or Cy5 (gene N), while the presence of a single positive signal on Fam channel was considered inconclusive, being the gene RdpR designed to be in common with other Sabercoviridae. On the contrary the absence of a signal on all channels, with the exclusion of Hex (that of β-actin), allowed us to conclude a sample as negative. The absence of a signal in all channels allowed us to conclude a sample as invalid due probable inhibition or an unreliable sampling. Table 1 reports the criteria of interpretation. Figure 1 reports the curve and the relative CT of a positive sample.
The results of our test of specificity and cross reactivity tests, performed using the ZeptoMetrix panels, showed the absence of any cross-reaction as well as the specific reaction of our primers and probes toward COVID-19 genes (Figure 2 ).
In total 166 samples were included in the study. 133 samples were negative, 31 resulted positive, two samples did not show amplification of endogenous internal control (β−actin) and were concluded as indeterminate.
Among 31 positive sample, six were positive for RdRp only, therefore reported as "inconclusive"; and 25 were COVID-19 positive. Among these 25, nine were positive for all the targets while 16
RdpR and E only (see Table 2 ).
The results showed by our assay were in agreement with those obtained using Novel Coronavirus All rights reserved. No reuse allowed without permission.
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Accurate and reliable diagnostic assays and large-scale testing are critical for early detection of a pathogen involved in an outbreak, but it is more crucial in case of an epidemic event for prompt public health actions. This has proven to be particularly true for SARS-CoV-2, which was identified as the cause of an outbreak of pneumonia, in Wuhan (China) in December 2019, and quickly spread all over in the world [15] [16] [17] [18] [19] [20] [21] . Laboratory diagnosis of infections caused by severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is mainly accomplished by performing nucleic acid amplification tests (NAATs) on respiratory tract specimens. Indeed, the upper respiratory tract specimens such as nasopharyngeal swabs and oropharyngeal swabs generally have high SARS-CoV-2 viral loads upon symptom onset [22] . Some authors have recently suggested to extend NAAT testing in order to include saliva and stool samples [23, 24] . Due to the pressing worldwide request of tests to diagnose COVID-19, numerous SARS-CoV-2 NAAT assays have become available and many others are in the final stages of development [20, 28, 29] . This aspect has the great advantage of making a wide range of diagnostic tests available to health systems and therefore allows us to respond to the diagnostic needs caused by the pandemic, but on the other hand this incredible quantity and difference between each kit poses problems on the validation process itself. An important restriction to validation qRT-PCR molecular assays for detection of SARS-CoV-2 is the availability of virus RNA standard and debate is still ongoing as whether or not to consider integral SARS-CoV-2 RNA full length as a safety level 3 biological hazard [19, 20] . If debate reaches consensus on marking SARS-CoV-2 as Level 3, its treatments could only be performed by laboratories with appropriate level 3 (BSL3) security measures, whose number is scarce thus limiting the capacity of carrying out experimental tests [28] . Taking into account these critical issues we have developed a qRT-PCR assay able to detect three target genes of SARS CoV-2 which strongest aspect is the characteristics of IC which is able to evaluate jointly the accurate collection of nasopharyngeal samples and the presence of any inhibitors in the PCR reaction. Sampling in SARS-CoV-2 is, in fact, one of the most frequent and probable cause of false negative results and therefore of delayed All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted June 19, 2020. . https://doi.org/10.1101/2020.06.17.20124396 doi: medRxiv preprint diagnosis [15, 28] . Moreover, in order to avoid to work with a full-lenght viral RNA, overcoming the issue of BSL3, we have chosen to construct artificial chimeric plasmid to test our primes and probes and at the same time to use ZeptoMetrix panels which allowed us to evaluate the specificity of our assay in safe conditions.
Our kit was designed to be open, either for nucleic acid extraction step or on the RT-PCR assay to be carried out on several instruments (in the present paper we tested two of them). Therefore, our assay can be used in any molecular biology laboratory. Furthermore, our kit is free from the industrial production logics of "closed systems" and conversely it is hypothetically available for distribution on large numbers. This aspect, at a time of great demand for tests and of equally known shortcomings of commercial kits, can be a significant strength that facilitates the introduction into microbiological laboratories.
In addition, taking into account potential genetic drift of SARS-CoV-2, especially as the virus evolves within new populations, and albeit literature suggests at least two molecular specific targets should be included in the assay to reduce the likely of cross-reactions, we are implementing our kit by adding a forth target gene codifying for glycoprotein spike (S) S gene, but validation tests are still in progress [25] [26] [27] [28] [29] . Finally, as evidences emerged in literature show that aside to direct respiratory sampling, rectal swab as well as saliva could be proper specimens to enhance the diagnosis of COVID-19, we are also extending validation test of our assay on such direction [28] . In presence of strong signal of others genes, the signal of IC may be inhibited but the results still valid.
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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted June 19, 2020. . https://doi.org/10.1101/2020.06.17.20124396 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted June 19, 2020. . https://doi.org/10.1101/2020.06.17.20124396 doi: medRxiv preprint Figure 2 . Results of qRT-PCR assays obtained using ZeptoMetrix TM panels in detecting the target genes of SARS-CoV-2 virus.
N RdpR E All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted June 19, 2020. . https://doi.org/10.1101/2020.06.17.20124396 doi: medRxiv preprint