Detection of Low-Copy Human Virus DNA upon Prolonged Formalin Fixation

Formalin fixation, albeit an outstanding method for morphological and molecular preservation, induces DNA damage and cross-linking, which can hinder nucleic acid screening. This is of particular concern in the detection of low-abundance targets, such as persistent DNA viruses. In the present study, we evaluated the analytical sensitivity of viral detection in lung, liver, and kidney specimens from four deceased individuals. The samples were either frozen or incubated in formalin (±paraffin embedding) for up to 10 days. We tested two DNA extraction protocols for the control of efficient yields and viral detections. We used short-amplicon qPCRs (63–159 nucleotides) to detect 11 DNA viruses, as well as hybridization capture of these plus 27 additional ones, followed by deep sequencing. We observed marginally higher ratios of amplifiable DNA and scantly higher viral genoprevalences in the samples extracted with the FFPE dedicated protocol. Based on the findings in the frozen samples, most viruses were detected regardless of the extended fixation times. False-negative calls, particularly by qPCR, correlated with low levels of viral DNA (<250 copies/million cells) and longer PCR amplicons (>150 base pairs). Our data suggest that low-copy viral DNAs can be satisfactorily investigated from FFPE specimens, and encourages further examination of historical materials.

Streamlined single-cell proteomics by an integrated microfluidic chip and data-independent acquisition mass spectrometry

Single-cell proteomics can reveal cellular phenotypic heterogeneity and cell-specific functional networks underlying biological processes. Here, we present a streamlined workflow combining microfluidic chips for all-in-one proteomic sample preparation and data-independent acquisition (DIA) mass spectrometry (MS) for proteomic analysis down to the single-cell level. The proteomics chips enable multiplexed and automated cell isolation/counting/imaging and sample processing in a single device. Combining chip-based sample handling with DIA-MS using project-specific mass spectral libraries, we profile on average ~1,500 protein groups across 20 single mammalian cells. Applying the chip-DIA workflow to profile the proteomes of adherent and non-adherent malignant cells, we cover a dynamic range of 5 orders of magnitude with good reproducibility and <16% missing values between runs. Taken together, the chip-DIA workflow offers all-in-one cell characterization, analytical sensitivity and robustness, and the option to add additional functionalities in the future, thus providing a basis for advanced single-cell proteomics applications.

Development of high-resolution melting (HRM) assay to differentiate the species of Shigella isolates from stool and food samples

Shigella species, a group of intracellular foodborne pathogens, are the main causes of bacillary dysentery and shigellosis in humans worldwide. It is essential to determine the species of Shigella in outbreaks and food safety surveillance systems. The available immunological and molecular methods for identifying Shigella species are relatively complicated, expensive and time-consuming. High resolution melting (HRM) assay is a rapid, cost-effective, and easy to perform PCR-based method that has recently been used for the differentiation of bacterial species. In this study, we designed and developed a PCR-HRM assay targeting rrsA gene to distinguish four species of 49 Shigella isolates from clinical and food samples and evaluated the sensitivity and specificity of the assay. The assay demonstrated a good analytical sensitivity with 0.01–0.1 ng of input DNA template and an analytical specificity of 100% to differentiate the Shigella species. The PCR-HRM assay also was able to identify the species of all 49 Shigella isolates from clinical and food samples correctly. Consequently, this rapid and user-friendly method demonstrated good sensitivity and specificity to differentiate species of the Shigella isolates from naturally contaminated samples and has the potential to be implemented in public health and food safety surveillance systems.

Development and Validation of a One-Step Reverse Transcription Real-Time PCR Assay for Simultaneous Detection and Identification of Tomato Mottle Mosaic Virus and Tomato Brown Rugose Fruit Virus

Tobamovirus species represent a threat to solanaceous crops worldwide, due to their extreme stability and being seed-borne. In particular, recent outbreaks of tomato brown rugose fruit virus in tomato and pepper crops led to the establishment of prompt control measures, and the need for reliable diagnosis was urged. Another member of the genus, tomato mottle mosaic virus, has recently risen attention due to reports in different continents and its common features with tomato brown rugose fruit virus. In this study, a new real-time RT-PCR detection system was developed for tomato brown rugose fruit virus and tomato mottle mosaic virus on tomato leaves and seeds using TaqMan chemistry. This test was designed to detect tomato mottle mosaic virus by amplifying the movement protein gene in a duplex assay with tomato brown rugose fruit virus target on the CP-3&rsquo;NTR region, which was already validated as a single assay. The performance of this tests was evaluated, displaying analytical sensitivity 10-5-10-6-fold dilution for seeds and leaves, respectively, and good analytical specificity, repeatability, and reproducibility. Using the newly developed and validated test, tomato brown rugose fruit virus detection was 100% concordant with previously performed analyses on 106 official samples collected in 2021 from different continents.

Reverse Transcription Recombinase-aided Amplification Assay for H5 Subtype avian Influenza Virus

Background: The H5 subtype avian influenza virus (AIV) has caused huge economic losses to the poultry industry and is a threat to human health. A rapid and simple test is needed to confirm infection in suspected cases during disease outbreaks. Methods: In this study, we developed a reverse transcription recombinase-aided amplification (RT-RAA) assay for the detection of H5 subtype AIV. Assays were performed at a single temperature (39°C), and the results were obtained within 20 min. Results: The assay showed no cross-detection with Newcastle disease virus or infectious bronchitis virus. The analytical sensitivity was 103 RNA copies/μL at a 95% confidence interval according to probit regression analysis, with 100% specificity. Compared with published reverse transcription quantitative real-time polymerase chain reaction assays, the κ value of the RT-RAA assay in 420 avian clinical samples was 0.983 (p < 0.001). The sensitivity for avian clinical sample detection was 97.26% (95% CI, 89.56–99.52%), and the specificity was 100% (95% CI, 98.64–100%). Conclusions: These results indicated that our RT-RAA assay may be a valuable tool for detecting H5 subtype AIV.

Analytical sensitivity of seven SARS-CoV-2 antigen-detecting rapid tests for Omicron variant

The emergence of novel SARS-CoV-2 variants of concern (VOCs) requires investigation of a potential impact on diagnostic performance, especially on Antigen-detecting rapid antigenic tests (Ag-RDT). Although anecdotal reports have been circulating that Omicron is in principle detected by several Ag-RDTs, no published data are a yet available for the newly emerged Omicron variant. Here, we have performed an analytical sensitivity testing with cultured virus in seven Ag-RDTs for their sensitivity to Omicron compared to data earlier obtained on VOCs Alpha, Beta Gamma and Delta and a pre-VOC isolate of SARS-CoV-2. Overall, we have found a tendency towards lower sensitivity for Omicron compared to pre-VOC SARS-CoV-2 and the other VOCs across tests. Importantly, while analytical testing with cultured virus may be a proxy for clinical sensitivity, is not a replacement for clinical evaluations which are urgently needed for Ag-RDT performance in Omicron-infected individuals.

Assessment of the analytical sensitivity of ten lateral flow devices against the SARS-CoV-2 omicron variant

Timely and accurate diagnostic testing is a critical component of the public health response to COVID-19. Antigen tests are used widely in many countries to provide rapid, economical and accessible point-of-care testing (1). The vast majority of antigen tests detect nucleocapsid (N) protein, a structural protein that displays less variation than the spike (S) protein across different SARS-CoV-2 lineages. Although antigen tests are less sensitive than RT-PCR tests, their ability to quickly detect individuals with high viral loads provides clinical and public health utility in many countries, including Australia, where antigen tests have recently been approved for self-testing (2). As new variants arise, including the recent emergence of the SARS-CoV-2 omicron variant, it is essential to rapidly assess the performance of diagnostic assays. Here, in order to assess and compare the ability of antigen tests to detect delta and omicron variants, we performed a rapid assessment of ten commercially available antigen tests.