The coronavirus disease 2019 (COVID-19) has caused a large global outbreak. It is accordingly important to develop accurate and rapid diagnostic methods. The polymerase chain reaction (PCR)-based method including reverse transcription-polymerase chain reaction (RT-PCR) is the most widely used assay for the detection of SARS-CoV-2 RNA. Along with the RT-PCR method, digital PCR has emerged as a powerful tool to quantify nucleic acid of the virus with high accuracy and sensitivity. Non-PCR based techniques such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA) are considered to be rapid and simple nucleic acid detection methods and were reviewed in this paper. Non-conventional molecular diagnostic methods including next-generation sequencing (NGS), CRISPR-based assays and nanotechnology are improving the accuracy and sensitivity of COVID-19 diagnosis. In this review, we also focus on standardization of SARS-CoV-2 nucleic acid testing and the activity of the National Metrology Institutes (NMIs) and highlight resources such as reference materials (RM) that provide the values of specified properties. Finally, we summarize the useful resources for convenient COVID-19 molecular diagnostics.

The human rhinovirus (RV) is a positive-stranded RNA virus that causes respiratory tract diseases affecting both the upper and lower halves of the respiratory system. RV enhances its replication by concentrating RNA synthesis within a modified host membrane in an intracellular compartment. RV infections often occur alongside infections caused by other respiratory viruses, and the RV virus may remain asymptomatic for extended periods. Alongside qualitative detection, it is essential to accurately quantify RV RNA from clinical samples to explore the relationships between RV viral load, infections caused by the virus, and the resulting symptoms observed in patients. A reference material (RM) is required for quality evaluation, the performance evaluation of molecular diagnostic products, and evaluation of antiviral agents in the laboratory. The preparation process for the RM involves creating an RV RNA mixture by combining RV viral RNA with RNA storage solution and matrix. The resulting RV RNA mixture is scaled up to a volume of 25 mL, then dispensed at 100 µL per vial and stored at −80 °C. The process of measuring the stability and homogeneity of RV RMs was conducted by employing reverse transcription droplet digital polymerase chain reaction (RT-ddPCR). Digital PCR is useful for the analysis of standards and can help to improve measurement compatibility: it represents the equivalence of a series of outcomes for reference materials and samples being analyzed when a few measurement procedures are employed, enabling objective comparisons between quantitative findings obtained through various experiments. The number of copies value represents a measured result of approximately 1.6 × 10⁵ copies/μL. The RM has about an 11% bottle-to-bottle homogeneity and shows stable results for 1 week at temperatures of 4 °C and −20 °C and for 12 months at a temperature of −80 °C. The developed RM can enhance the dependability of RV molecular tests by providing a precise reference value for the absolute copy number of a viral target gene. Additionally, it can serve as a reference for diverse studies.

Severe fever with thrombocytopenia syndrome (SFTS) is caused by SFTS virus (SFTSV) and is characterized by high mortality and non-specific clinical manifestations, which make early and accurate molecular diagnosis essential. This study aims to investigate whether reverse transcription droplet digital PCR (RT-ddPCR) provides improved diagnosis of SFTS compared to existing molecular diagnostic platforms. Detection limits of RT-ddPCR, reverse transcription quantitative PCR (RT-qPCR), and reverse transcription nested PCR (RT-nested PCR) were determined using RNA extracted from metrologically validated SFTSV nucleic acid reference material. Then, diagnostic performance metrics of each PCR method were compared using RNA from 29 SFTS patients and 29 disease controls with scrub typhus. Statistical analyses of key laboratory parameters were performed to identify markers significantly correlated with SFTSV load, as defined by RNA copy number, in early SFTS patients. RT-ddPCR showed the lowest detection limit of 3.82 copies/μL among the three PCR methods. The sensitivity of RT-ddPCR was 82.75% in clinical samples, which was higher than that of RT-qPCR at 72.41%. The specificity of RT-ddPCR was 100% compared to 89.66% for RT-nested PCR. Overall, RT-ddPCR demonstrated the highest diagnostic performance among the three PCR methods, with an accuracy of 91.38% and an area under the curve value of 0.91. A strong correlation between viral RNA loads and laboratory parameters was observed in the early stage of SFTS, particularly with high-sensitivity C-reactive protein, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and activated partial thromboplastin time. Our findings demonstrate that a novel molecular diagnosis method using RT-ddPCR shows improved diagnostic performance for detecting SFTSV RNA from SFTS patients and potentially contributes to accurate early diagnosis and enhanced clinical diagnosis. IMPORTANCE: Severe fever with thrombocytopenia syndrome (SFTS) is a high-mortality tick-borne infection caused by SFTS virus (SFTSV), and reliable molecular diagnosis is essential for patient care in endemic regions. Existing PCR-based assays for SFTS show limited sensitivity and specificity, particularly in patients with suspected or early-stage disease. Here, we describe the development and analytical validation of a reverse transcription droplet digital PCR (RT-ddPCR) assay for the detection and quantification of SFTSV RNA from patient sera. By using metrologically validated viral nucleic acid reference materials and clinical samples, this assay demonstrates significantly improved sensitivity and overall diagnostic performance compared with conventional reverse transcription quantitative PCR and reverse transcription nested PCR. RT-ddPCR can provide clinical microbiology laboratories with a useful tool to enhance early and reliable molecular diagnosis of SFTS.

Rift Valley fever virus (RVFV) has mainly been reported in African countries, and molecular diagnosis is performed using nucleic acid amplification tests.