The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become an important challenge worldwide. Widespread and strategic testing has been one of the most important majors taken to control the spread of the disease [1,2]. In addition to the COVID-19 pandemic, other respiratory pathogens like influenza and respiratory syncytial virus (RSV) are circulating in the population. Although the transmission of these respiratory viruses had been decreased because of the protective measures against SARS-CoV-2 [3], due to the increasing rate of vaccination, lockdown discontinuance and relaxing on mask-wearing [4] these respiratory viruses are rising again, causing a threat of a ‘triplepidemic’ [5,6] Influenza and RSV have some similar clinical features compared to COVID-19 [7,8] Additionally, some co-infections with SARS-CoV-2 are also reported [9], [10], [11], [12]. Therefore, multiplex detection of SARS-CoV-2, influenza A/B, and RSV might be needed for discriminating between these viruses and using specific treatments.

Real-time reverse transcription-polymerase chain reaction (rRT-PCR) is known as the gold standard for SARS-CoV-2 [13], influenza A/B [14], and RSV [15] detection. Before performing rRT-PCR, the sample goes through sample preparation, including lysis of the virus particles and isolation of the total nucleic acid from the sample [16]. Nucleic acid extraction is performed to remove the potential contaminant of the biological sample, which can inhibit the function of the DNA polymerase [17]. However, this step is time-consuming, labour-intensive, and possible to introduce other human errors to the whole process due to the increased technician interventions. Skipping the extraction step would eliminate the need for extraction kits, decrease the reagent consumption and lower the per-test price. Additionally, it can scale up the lab throughput by reducing the time, and enable meeting a higher number of tests per day [18,19]. Several studies have examined bypassing nucleic acid extraction and using only thermal lysis as the sample preparation procedure [18,[20], [21], [22], [23], [24], [25]].

Lab-on-a-chip (LOC) platforms use micro/nanofabrication methods to construct micro/nanochannels and fluidic unit operations for handling fluid on a micro/nano scale [26]. These platforms have been widely used in analytical applications, especially point-of-care test (POCT) devices [27] due to advantages such as integration of all steps on a microfluidic chip, automation, multiplexing, high throughput analysis, and cost-effectiveness (because of lower reagent consumption) [28,29]. POCTs have been conventionally limited to glucose sensors for diabetes control or lateral-flow immunoassays for at-home pregnancy tests [27]. However, there is a trend toward bringing more sophisticated assays such as molecular diagnostics to the point of care by benefiting from special features that LOC platforms can provide. [30] In particular, there has been a wide effort to implement molecular diagnostic (e.g., PCR) [31] and required sample preparation (i.e., lysis and nucleic acid extraction) [32,33], on a microfluidic chip to make an integrated, automated, and sample-to-answer POCT device for infectious disease diagnostics. To lower the complexity and cost of the microfluidic chip, there have been studies on bypassing the nucleic acid isolation step and designing an extraction-free PCR chip [34,35]

In a previous study conducted by our research group, an innovative strategy was introduced to design and develop a device and a disposable microfluidic chip for performing rRT-PCR for detecting SARS-CoV-2 [36]. This advancement holds considerable promise by improving on-chip heat transfer to the PCR mixture leading to enhanced turn-around time and accuracy. This approach will be more efficient when integrated with a simplified, cost-effective, and fast sample preparation protocol. In this study, we aim at evaluating an extraction-free sample preparation method using heat treatment for multiplex detection of RSV, influenza A/B, and SARS-CoV-2. Next, we try to implement this extraction-free method on a microfluidic chip, with the perspective of designing an extraction-free rRT-PCR chip (Scheme 1).