The res4plex direct RT-PCR is a real-time RT-qPCR test for simultaneous in vitro detection and differentiation of RNA from SARS-CoV-2, influenza A, influenza B, and RSV A/B. The res4plex direct RT-PCR test was validated with the Roche LightCycler® 480 II and with the BioRad CFX Opus 96™. In general, Lab direct PCR tests are compatible with other qPCR cyclers (e.g., MIC Cycler, AJ qTOWER, LightCycler® 96 or 480 I).

1. Name of the device res4plex direct RT-PCR 2. Intended Purpose The res4plex direct RT-PCR test is an assay for in vitro examination of viral RNA in nasal, nasopharyngeal or oral/oropharyngeal swabs to provide information to aid to diagnose symptomatic or asymptomatic patients under suspicion of respiratory diseases: SARS-CoV-2, influenza A, influenza B, and respiratory syncytial virus A/B (RSV A/B). The IVD medical device detects the RNA of the aforementioned pathogens by qualitative measurements based on RT-qPCR and is intended for use in medical laboratories or health institutions by laboratory...

Symptoms typical of influenza are characterized by sudden illness, fever, cough or sore throat, and muscle aches and/or headache. Other symptoms may include general weakness, sweating, rhinorrhea, and rarely nausea/vomiting and diarrhea [8]. SARS-CoV-2 is classified within the genus Betacoronavirus (subgenus Sarbecovirus) of the family Coronaviridae [9]. Betacoronaviruses also include SARS-CoV, MERS-CoV (Middle East respiratory syndrome coronavirus), and the human coronaviruses (HCoV) HKU1 and OC43 that circulate as cold viruses [10]. Infections with SARS-CoV-2 lead to a new disease profile called...

Coronaviruses are zoonotic, meaning they are transmitted between animals and humans. Common signs of infection include respiratory symptoms, fever, cough, shortness of breath and breathing difficulties. In more severe cases, infection can cause pneumonia, severe acute respiratory syndrome, kidney failure and even death [18]. Respiratory Syncytial Virus (RSV), discovered in 1956, is one of the most common causes of childhood illness [19]. It is a single-stranded (ss), negatively oriented (-), unsegmented RNA virus of the family Pneumoviridae (genus Orthopneumovirus) (see Figure 3). It has a bilayer...

The res4plex direct RT-PCR test is a multiplex RT-qPCR test for the detection and differentiation of SARS-CoV-2, influenza A, influenza B and RSV (A&B). A separate full process run control (Internal Control; IC) is added to each sample prior to RNA extraction and serves as control for nucleic acid isolation from the biological specimen as well as for RT-qPCR. The RNA eluate is added to the ready-to-use reaction solution that contains all reagents necessary for RT-qPCR. RT-qPCR analysis can be performed on various RT-qPCR cyclers. The res4plex direct RT-PCR test contains primer and probes specific...

The res4plex direct RT-PCR kit is available in the following variants: Table 4: res4plex direct RT-PCR configurations • qPCR cycler* • Disposable protective gloves, powder-free • PCR reaction tubes/microtiter plate plus lids/adhesive optical film • sterile filter-tips for PCR testing (DNA/RNA- and RNase-free) • Table centrifuge • Negative Control (no-template control, molecular grade water, or any other negative control according to the laboratory's standard procedure) *The res4plex direct RT-PCR test was validated with the Roche LightCycler® 480 II and with the BioRad CFX Opus 96™. In general,...

> Discard sample and assay waste (that was in contact with patient material) according to your local, regional, or national safety regulations. > The concentration specifications and incubation times of the manufacturers must be followed. > Do not use the test beyond the expiration date. > Do not use the test with opened or damaged packaging. > Protect reagents from heat, moisture, and light. > Do not replace or mix the reagents with reagents from other batches or other chemicals. > Avoid contamination of the test by microorganisms and nucleases (DNases and RNases). > Any carry-over of samples...

This test is intended for use with samples derived from nasal, nasopharyngeal or oral/oropharyngeal. Other specimen types may cause invalid results. In every RT-qPCR run one Positive Control and one Negative Control should be included. The Positive Control consists of nucleic acids of SARS-CoV-2, influenza A, influenza B and RSV. The Negative Control (e.g. molecular grade water) must be provided by the user. A failed Positive or Negative Control will invalidate the RT-qPCR run and the results must not be reported. Appropriate nucleic acid extraction methods must be conducted prior to using this...

The res4plex direct RT-PCR test was validated with the Roche LightCycler® 480 II and BioRad CFX Opus 96™. In general, FRIZ Lab direct PCR tests are compatible with many RT-qPCR cyclers. The following thermal profile is to be used (see Table 6). Channel settings depend on the RT-qPCR cycler used and should be checked before starting the analysis (see Table 7 and Table 8). FBC107-Ax is suitable for RT-qPCR cyclers with at least the five channels Cyan 500, FAM, HEX, Red 610, and Cy5 (e.g., LightCycler® 480 II), FBC107-Bx is suitable for RT-qPCR cyclers with at least the five channels FAM, HEX, Red...

14. Results Positive samples show a qPCR typical amplification curve that crosses a certain threshold generating the Ct value (see Figure 5). For distribution of results within the channels see Table 9 and Table 10. A positive signal is characterized by a sigmoidal curve exhibiting both a lag phase and a logarithmic growth phase. Signals that do not exhibit this curve pattern, despite having a Ct value, are considered negative. The threshold shall be set within the logarithmic growth phase. It should be noted that in the case of low positive samples, the stationary phase might not be visible...

Table 9: Results of res4plex direct RT-PCR (Ref. No.: FBC107-Ax and FBC107-Bx) 15. Limitations of the Method Test results should always be seen in the context of the clinical findings. Therapeutic consequences of the diagnostic results must be drawn in relation to the clinical findings. The detection of analyte target does not mean that they are the causative agents of clinical symptoms. Mutations or polymorphisms in primer and probe binding regions can interfere with the detection of new variants that may result in false negative results. Human blood is a PCR inhibitor [27] and was found to...