MicroFLOQ® Direct: A Helpful Tool for the Coronavirus SARS-Cov-2 Rapid Detection without RNA Purification

In the context of SARS-Cov-2 virus disease (Covid19) pandemic, molecular diagnostic tools were rapidly developed as there are fundamental for a rapid detection of infected people. In this context, and in order to optimize the manipulations and reduce the time to get results, we report the successful use of a sampling tool for Covid19 diagnosis named microFLOQ® Direct (MFD). Hundred upper respiratory specimens sampled from patients with potential Covid19 were evaluated using MFD, and results were compared to the results obtained by standard sampling procedure using dry swabs and physiologic serum as the transport medium. MFD results compared to results issued from the classic RNA purification and amplification steps from transport medium showed that MFD can be directly used for RT-PCR analysis without the preliminary inactivation and extraction steps. So, MFD could limit handling errors compared to the different treatment steps with dry swabs and transport medium, and therefore the risk of operator contamination, and could simplify the analytical process and enables to get results in less than 2 hours. We expect that the proposed detection kit using MFD sampling represents a relevant and operational screening tool in the field of molecular detection of viral and bacterial diseases during pandemic or for public health or agro-veterinary purposes. Human RNA purification PCR reaction to residual inhibitors after purification, while a human CT value 26 with microFLOQ® Direct. These results the limit the sensibility of the method but, on the limit the impact of residual after


Context
Since the March 24, 2020, French public authorities as well as the whole world state a health crisis caused by a novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) named COVID-19, and described for the first time in December 2019 in China [1]. This global health crisis highlights the need for a mobile diagnostic force at national and international level, with clinical diagnostic laboratories to meet the quality standards for medical biology (ISO15189) [2], to best address the needs.
The French National Gendarmerie, as an interior security forces serving the protection of the French people, has taken the initiative to adapt all their possibilities and, moreover, to adapt the forensic Mobil'DNA laboratory [3] as the mobile Covid19 diagnostic force. The objective is to propose an autonomous mobile biomolecular laboratory for rapid tests at high throughput level for the endemic pathogen COVID-19, in support of local hospitals and health systems, in rural or confined areas.
Backed as a proof of concept to pandemic COVID-19, the proposed scheme could be projected in the future in any type of pandemic in areas involving rapid and autonomous reliable detection such as public health, agro-veterinary, plant health screening. In order to be efficient for this mobile force for microbiological diagnosis, the Forensic research institute of from the Gendarmerie Nationale (IRCGN) combined its expertise in mobile and high-speed analysis of biological samples with the expertise of a French medical microbiology laboratory in the diagnosis of endemic pathogens [3].
In order to optimize the diagnostic work in forensic, IRCGN has patented (WO2016132028A1) an original DNA sampling tool [4], the microFLOQ® Direct swab (MFD) (see additional figure 1a) in order to make forensic DNA tests easier, faster and directly on the field [5]. Manufactured by COPAN Company (Italy) and initially developed for forensic DNA analysis for crime scene or disaster victim identification (DVI) context, the MFD swab is a miniaturized version of floq swab that presents a 1 mm2 swab head and a breaking point (see additional figure 1b) that fit perfectly with microtube or with a 96 well PCR microplate. Flocked fibres to the head of the swab present high affinity for nucleic acids and are embedded by lysing agents which allows direct amplification and DNA analysis from sample collection to a final result in less than two hours. Additionally, the MFD swab subsamples only a minute portion of the biological material (2 µl) and preserves the vast majority of the sample for subsequent testing or reanalysis. By these properties MFD swab is a useful and smart device for rapid molecular screening on coronavirus SARS-CoV-2. In this article, we described a study carried out on 100 samples in order to make a correlation and show that the RT-PCR analysis of COVID-19 can skip the RNA extraction step by simply using the microFLOQ® Direct to detect SARS-CoV-2.

The genetic diagnosis of SARS-CoV-2
The SARS-CoV-2 detection is performed from purified RNA by reverse-transcriptase PCR (RT-PCR) [6][7][8]. The RT-PCR is performed with the GeneFirst RUO COVD-19 Detection Kit (genefirst®; UK) that was validated by the French National Reference Centre for respiratory viruses including flu viruses (Pasteur Institute, Paris, France) following the extraction by the Ademtech extraction kit (Ademtech, Bordeaux, France). GeneFirst RT-PCR allowed detection of two specific targets of COVID-19 (N Gene and ORF1 corresponding to the polymerase gene) and one internal control (GAPDH human target) [6,9]. Amplification was performed using Applied™ 7500 Real Time PCR system from ThermoFisher (software v2.3). Three different controls were used to define the base lines and the threshold to be applied to unknown samples. The RNA extraction negative control is composed of nuclease free water. The Internal Quality control (ICQ) is composed of quantified virus RNA from positive patients COVID-19 with a defined Ct. The Ct value expected for this ICQ was defined as between 25 and 27. The Positive control is from the supplier (GeneFirst® RUO COVID-19 Detection kit).

Sample Collection
One hundred samples were collected from symptomatic and asymptomatic patients, present in different institutions (nursing homes, prisons, barracks, etc.). The samples were collected using nasopharyngeal swabs from Copan® according to the manufacturer's recommendations (14). Samples were processed within 24 hours after collection otherwise stored during at +4°C before being processed. All the samples were processed at the same time firstly with microFLOQ® Direct and secondly with standard RNA purification method. 250 µL from 1000 µL of transport medium is used for RNA purification. For swabs with alginate conservative medium such as the Copan, total head was discharged in the lysis buffer. RNA purification was performed by lysis of the clinical sample using chemical agents followed by magnetic beads purification using the viral prep Adem-kit® (Ademtech, France), according to the manufacturer recommendations. The extraction step was processed on a KingFisher Flex 96 (ThermoFisher) allowing 96 purifications in 30 minutes. Interpretation and the generation of reports were carried out using GendLink® software adapted to generate a report on the results of COVID-19

Sample processing and sample preparation for amplification and SARS-CoV
analyses.

Sample Processing Using microFLOQ® Direct Swab
The MFD cannot be used directly for nasopharyngeal sampling because the tip is too short to reach the nasal cavity, and with a high risk of breaking inside the cavity. Furthermore, the lysis agents embedded on the fibres are also not safe for humans. In this study, MFD swab was used by subsampling the regular nasopharyngeal swab after collection.
For subsampling, the MFD swab was brought into contact with the tip of the swab used for nasopharyngeal sampling 2 seconds by tapping (see additional figure 2). In the absence of a swab in the sample (swab discharged into the transport medium), the MFD swab is brought into contact for 2 seconds with the transport medium present in the cap of the sample tube after shaking the tube (see additional figure 3). MFD was then broken at his breakable point in the well of a 96 well PCR micro plate or in a micro tube containing 10 μl of elution buffer (Nuclease Free water). The elution step occurred during 10 minutes, at room temperature, under stirring at 500 rpm. A 5 μl of elution buffer was taken and added to 15 μl of RT-PCR mix to then be amplified.

Interpretation of results
There

Concordance study
For each COVID +, COVIDand COVID undetermined populations and categories, we compared the results obtained with conventional RNA purification protocol to those obtained from microFLOQ® Direct protocol in order to determine the concordance or discordance between the two methods.
Concerning patients declared non-infected by COVID-19 using regular method, a complete concordance is obtained for all the samples (n=57

Discussion
The devices and processes for the collection of biological materials favouring a rapid and simplified DNA analysis each respond to a particular problem, but none meets a global need: having the same and the most simplified tool to collect and analyse as quickly as possible DNA and RNA [10].
In addition, the devices must not alter the biological sample in order to authorize additional analyses and must secure the biological material collected to minimize the risk of contamination. They must also be compatible with conventional methods, reagents and analytical instruments commonly used in analysis laboratories, particularly compatible with RT-PCR amplification.
Finally, they must be compatible with high throughput processing of samples without the need for robotic sample preparation platforms, have efficiency at least equivalent to that of current devices and if possible reduce costs compared to the cost of a conventional analysis.
The microFLOQ® Direct swab is a device used in the proposed alternative process, easy to use and compatible for high-speed analyses without the need for nucleic acids purification reagent reactions and instrument. The presentation of these comparative results on 100 samples between the conventional RNA analysis method and the alternative method using microFLOQ® Direct swab shows that microFLOQ® Direct can be used to carry out tests for RT-PCR detection of SARS-CoV-2.
Subsampling immediately with the microFLOQ® Direct swab the regular swab used for the sample collection could be a technical and logistical advantage if we can demonstrate that the lysis agents embedded on the fibres microFLOQ® Direct swab inactivates instantly the virus. In this way, it is therefore no longer compulsory to work in the laboratory under type 2 microbiological safety cab (MSC). The CT shift observed between microFLOQ® In order to enhance the quantity of the virus to be analysed without RNA purification, we could propose the use of FTA cards instead of microFLOQ® Direct swab. FTA card is also treated with lysis reagents allowing direct RNA amplification [11]. The benefit to the FTA card is the possibility to perform multiples punching at different diameters in order to enhance the quantity of samples analysed [12].
However, if you need to process a lot of samples, the use of the semi-automatic or automatic puncher with a unique punching head is a putative risk of cross contamination that must be evaluated. To prevent this risk, the best way could be to change the punching head between each sample or clean the punching head that is time consuming, not cost effective and not compatible with high throughput screening. Even if the quantity of samples collected is a limitation for the sensibility of the method, the one shot use of microFLOQ® Direct swab could be the best compromise for rapid screening by preventing the risk of cross contamination during the analytical process.

Conclusıon
On 100 samples, we found that the microFLOQ® Direct swab presents an interesting alternative to conventional nucleic acid purification method to analyse viruses like coronavirus SARS-Cov-2. During an active epidemic phase, microFLOQ® Direct swab could be an innovative strategy for rapid screening test to detect viruses in less than 90 minutes and adopt immediately the measures to stop the propagation of the virus. By consuming an insignificant fraction of the sample, the subsampling method using microFLOQ® Direct swab enables to store the original sample (nasopharyngeal swab or liquid) in order to build a bio bank very useful for technical and clinical research.
In addition, the use of microFLOQ® Direct swab reduce analysis costs compared to the conventional RNA purification reagents and instruments, reducing the working time and handling steps while ensuring greater protection for the manipulator and offer the possibility to perform the analyse directly to the collection site inside a mobile laboratory.