MassARRAY: a high-throughput solution for rapid detection of foodborne pathogens in real-world settings.

Introduction: Bacterial foodborne pathogens pose a substantial global public health concern, prompting government agencies and public health organizations to establish food safety guidelines and regulations aimed at mitigating the risk of foodborne illness. The advent of DNA-based amplification coupled with mass spectrometry, known as MassARRAY analysis, has proven to be a highly precise, sensitive, high-throughput, and cost-effective method for bacterial detection. This study aimed to develop, validate, and evaluate a MassARRAY-based assay for the detection and identification of significant enteropathogenic bacteria. Methods: The MassARRAY-based assay was developed for the detection of 10 crucial bacterial foodborne pathogens, including Campylobacter coli, Campylobacter jejuni, Clostridium perfringens, Escherichia coli, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Salmonella spp., Shigella spp., and Staphylococcus aureus. The assay was optimized using the reference gDNA (n = 19), followed by validation using gDNA (n = 85) of reference and laboratory isolates. Additionally, the evaluation of the assay's reaction using a mixture of gDNA from all nine targeted species was performed. The limit of detection of the developed MassARRAY-based assay was determined using bacterial cells. Moreover, the validation method for field samples was evaluated by comparing it with standard microbiological testing methods routinely analyzed. Results: The developed MassARRAY-based assay demonstrated 100% concordance with known bacterial pure cultures. The assay's reaction using a mixture of gDNA from all nine targeted species revealed the MassARRAY's capability to detect all targeted species in a single assay with the lowest concentration of 1 ng/µL of gDNA. The limits of detection of the assay range from 357 ± 101 to 282,000 ± 79,196 cells. Moreover, the validation of the assay in field samples revealed a 100% correlation between the data obtained from the standard microbiological method and the MassARRAY-based assay. Discussion: These findings suggested that the developed MassARRAY-based assay exhibited the excellence in high-throughput detection of foodborne bacterial pathogens with high accuracy, reliability, and potential applicability within real-world field samples.

Comparative Performance in the Detection of Four Coronavirus Genera from Human, Animal, and Environmental Specimens.

Emerging coronaviruses (CoVs) are understood to cause critical human and domestic animal diseases; the spillover from wildlife reservoirs can result in mild and severe respiratory illness in humans and domestic animals and can spread more readily in these naïve hosts. A low-cost CoV molecular method that can detect a variety of CoVs from humans, animals, and environmental specimens is an initial step to ensure the early identification of known and new viruses. We examine a collection of 50 human, 46 wastewater, 28 bat, and 17 avian archived specimens using 3 published pan-CoV PCR assays called Q-, W-, and X-CoV PCR, to compare the performance of each assay against four CoV genera. X-CoV PCR can detect all four CoV genera, but Q- and W-CoV PCR failed to detect δ-CoV. In total, 21 (42.0%), 9 (18.0%), and 21 (42.0%) of 50 human specimens and 30 (65.22%), 6 (13.04%), and 27 (58.70%) of 46 wastewater specimens were detected using Q-, W-, and X-CoV PCR assays, respectively. The X-CoV PCR assay has a comparable sensitivity to Q-CoV PCR in bat CoV detection. Combining Q- and X-CoV PCR assays can increase sensitivity and avoid false negative results in the early detection of novel CoVs.

High Transmission Rates of Early Omicron Subvariant BA.2 in Bangkok, Thailand.

The emergence of Omicron as the fifth variant of concern within the SARS-CoV-2 pandemic in late 2021, characterized by its rapid transmission and distinct spike gene mutations, underscored the pressing need for cost-effective and efficient methods to detect viral variants, especially given their evolving nature. This study sought to address this need by assessing the effectiveness of two SARS-CoV-2 variant classification platforms based on RT-PCR and mass spectrometry. The primary aim was to differentiate between Delta, Omicron BA.1, and Omicron BA.2 variants using 618 COVID-19-positive samples collected from Bangkok patients between November 2011 and March 2022. The analysis revealed that both BA.1 and BA.2 variants exhibited significantly higher transmission rates, up to 2-3 times, when compared to the Delta variant. This research presents a cost-efficient approach to virus surveillance, enabling a quantitative evaluation of variant-specific public health implications, crucial for informing and adapting public health strategies.

A customized mass array panel for BCR::ABL1 tyrosine kinase domain mutation screening in chronic myeloid leukemia.

Introduction: The therapeutic strategy and management of chronic myeloid leukemia (CML) have rapidly improved with the discovery of effective tyrosine kinase inhibitors (TKIs) to target BCR::ABL1 oncoprotein. However, nearly 30% of patients develop TKI resistance due to acquired mutations on the tyrosine kinase domain (TKD) of BCR::ABL1. Methods: We customized a mass array panel initially intended to detect and monitor the mutational burden of hotspot BCR::ABL1 TKD mutations accumulated in our database, including key mutations recently recommended by European LeukemiaNet. Additionally, we extended the feasibility of using the assay panel for the molecular classification of myeloproliferative neoplasms (MPNs) by incorporating primer sets specific for analyzing JAK2 V617F, MPL 515 K/L, and CALR types 1 and 2. Results: We found that the developed mass array panel was superior for detecting and monitoring clinically significant BCR::ABL1 TKD mutations, especially in cases with low mutational burden and harboring compound/polyclonal mutations, compared with direct sequencing. Moreover, our customized mass array panel detected common genetic alterations in MPNs, and the findings were consistent with those of other comparable assays available in our laboratory. Conclusions: Our customized mass array panel was practicably used as a routine robust assay for screening and monitoring BCR::ABL1 TKD mutations in patients with CML undergoing TKI treatment and feasible for analyzing common genetic mutations in MPNs.

SARS-CoV-2 Variants Detection Strategies in Wastewater Samples Collected in the Bangkok Metropolitan Region.

Wastewater surveillance is considered a promising approach for COVID-19 surveillance in communities. In this study, we collected wastewater samples between November 2020 and February 2022 from twenty-three sites in the Bangkok Metropolitan Region to detect the presence of SARS-CoV-2 and its variants for comparison to standard clinical sampling. A total of 215 wastewater samples were collected and tested for SARS-CoV-2 RNA by real-time PCR with three targeted genes (N, E, and ORF1ab); 102 samples were positive (42.5%). The SARS-CoV-2 variants were determined by a multiplex PCR MassARRAY assay to distinguish four SARS-CoV-2 variants, including Alpha, Beta, Delta, and Omicron. Multiple variants of Alpha-Delta and Delta-Omicron were detected in the wastewater samples in July 2021 and January 2022, respectively. These wastewater variant results mirrored the country data from clinical specimens deposited in GISAID. Our results demonstrated that wastewater surveillance using multiple signature mutation sites for SARS-CoV-2 variant detection is an appropriate strategy to monitor the presence of SARS-CoV-2 variants in the community at a low cost and with rapid turn-around time. However, it is essential to note that sequencing surveillance of wastewater samples should be considered complementary to whole genome sequencing of clinical samples to detect novel variants.

Simultaneous detection of omicron and other SARS-CoV-2 variants by multiplex PCR MassARRAY technology.

The rapid emergence of SARS-CoV-2 variants with high severity and transmutability adds further urgency for rapid and multiplex molecular testing to identify the variants. A nucleotide matrix-assisted laser-desorption-ionization time-of-flight mass spectrophotometry (MALDI-TOF MS)-based assay was developed (called point mutation array, PMA) to identify four major SARS-CoV-2 variants of concern (VOCs) including Alpha, Beta, Delta, and Omicron (namely PMA-ABDO) and differentiate Omicron subvariant (namely PMA-Omicron). PMA-ABDO and PMA-Omicron consist of 24 and 28 mutation sites of the spike gene. Both PMA panels specifically identified VOCs with as low as 10 viral copies/µl. The panel has shown a 100% concordant with the Next Generation Sequencing (NGS) results testing on 256 clinical specimens with real-time PCR cycle threshold (Ct) values less than 26. It showed a higher sensitivity over NGS; 25/28 samples were positive by PMA but not NGS in the clinical samples with PCR Ct higher than 26. Due to the mass of nucleotide used to differentiate between wild-type and mutation strains, the co-infection or recombination of multiple variants can be determined by the PMA method. This method is flexible in adding a new primer set to identify a new emerging mutation site among the current circulating VOCs and the turnaround time is less than 8 h. However, the spike gene sequencing or NGS retains the advantage of detecting newly emerged variants.

Simultaneous Detection of Omicron and Other SARS-CoV-2 Variants by Multiplex PCR MassARRAY Technology.

The rapid emergence of SARS-CoV-2 variants with high severity and transmutability adds further urgency for rapid and multiplex molecular testing to identify the variants. A nucleotide matrix-assisted laser-desorption-ionization time-of-flight mass spectrophotometry (MALDI-TOF MS)-based assay was developed (called point mutation array, PMA) to identify four major SARS-CoV-2 variants of concern (VOCs) including Alpha, Beta, Delta, and Omicron (namely PMA-ABDO) and differentiate Omicron subvariant (namely PMA-Omicron). PMA-ABDO and PMA-Omicron consist of 24 and 28 mutation sites of the spike gene. Both PMA panels specifically identified VOCs with as low as 10 viral copies/ µl. The panel has shown a 100% concordant with the Next Generation Sequencing (NGS) results testing on 256 clinical specimens with real-time PCR cycle threshold (Ct) values less than 26. It showed a higher sensitivity over NGS; 25/28 samples were positive by PMA but not NGS in the clinical samples with PCR Ct higher than 26. Due to the mass of nucleotide used to differentiate between wild-type and mutation strains, the co-infection or recombination of multiple variants can be determined by the PMA method. This method is flexible in adding a new primer set to identify a new emerging mutation site among the current circulating VOCs and the turnaround time is less than 8 hours. However, the spike gene sequencing or NGS retains the advantage of detecting newly emerged variants.