Highly Specific Loop-Mediated Isothermal Amplification Using Graphene Oxide–Gold Nanoparticles Nanocomposite for Foot-and-Mouth Disease Virus Detection

Loop-mediated isothermal amplification (LAMP) is a molecular diagnosis technology with the advantages of rapid results, isothermal reaction conditions, and high sensitivity. However, this diagnostic system often produces false positive results due to a high rate of non-specific reactions caused by formation of hairpin structures, self-dimers, and mismatched hybridization. The non-specific signals can be due to primers used in the methods because the utilization of multiple LAMP primers increases the possibility of self-annealing of primers or mismatches between primers and templates. In this study, we report a nanomaterial-assisted LAMP method that uses a graphene oxide–gold nanoparticles (AuNPs@GO) nanocomposite to enable the detection of foot-and-mouth disease virus (FMDV) with high sensitivity and specificity. Foot-and-mouth disease (FMD) is a highly contagious and deadly disease in cloven-hoofed animals; hence, a rapid, sensitive, and specific detection method is necessary. The proposed approach exhibited high sensitivity and successful reduction of non-specific signals compared to the traditionally established LAMP assays. Additionally, a mechanism study revealed that these results arose from the adsorption of single-stranded DNA on AuNPs@GO nanocomposite. Thus, AuNPs@GO nanocomposite is demonstrated to be a promising additive in the LAMP system to achieve highly sensitive and specific detection of diverse diseases, including FMD.

Rapid and specific detection of intact viral particles using functionalized microslit silicon membranes as a fouling-based sensor

The COVID-19 pandemic demonstrated the public health benefits of reliable and accessible point-of-care (POC) diagnostic tests for viral infections. Despite the rapid development of gold-standard reverse transcription polymerase chain reaction...

Development and Validation of a Novel ELISA for the Specific Detection of Antibodies against Mycobacterium avium Subspecies paratuberculosis Based on a Chimeric Polyprotein

Bovine paratuberculosis (PTB) is caused by Mycobacterium avium subsp. paratuberculosis (MAP). The optimization of detection tests specific for MAP is crucial to improve PTB control. In this work, we aimed to develop and validate a diagnostic tool based on an ELISA to specifically detect anti-MAP antibodies from bovine serum samples. For that purpose, we designed a recombinant polyprotein containing four specific antigens from MAP and optimized the ELISA. The validation consisted of the assessment of 10 sera from PTB-infected and healthy bovines with different OD values. The diagnostic performance of the polyprotein-ELISA was evaluated by testing 130 bovine serum samples (47 healthy, 48 MAP-infected, and 35 M. bovis-infected bovines). The ELISA using the polyprotein yielded an area under the ROC curve (AUC) of 0.9912 (95% CI, 0.9758–1.007; P < 0.0001). Moreover, for this ELISA, the cut-off selected from the ROC curve based on the point with a sensitivity of 95.56% (95% CI, 0.8485–0.9946) and specificity of 97.92 (95% CI, 0.8893–0.9995) was 0.3328. Similar results were obtained with an ELISA using the commercial Paratuberculosis Protoplasmatic Antigen (PPA). However, the ELISA with the polyprotein antigen showed a better performance against sera from animals infected with Mycobacterium bovis compared to the ELISA with PPA: lower cross-reactivity (2.85% versus 25.71%). These results demonstrate a very low cross-reactivity of the polyprotein with antibodies present in serum samples from animals infected with M. bovis. The designed polyprotein and the validated ELISA could be very useful for the specific identification of MAP-infected animals in herds.

Development and comparison of loop-mediated isothermal amplification with quantitative PCR for the specific detection of Saprolegnia spp.

Saprolegniasis is an important disease in freshwater aquaculture, and is associated with oomycete pathogens in the genus Saprolegnia. Early detection of significant levels of Saprolegnia spp. pathogens would allow informed decisions for treatment which could significantly reduce losses. This study is the first to report the development of loop-mediated isothermal amplification (LAMP) for the detection of Saprolegnia spp. and compares it with quantitative PCR (qPCR). The developed protocols targeted the internal transcribed spacer (ITS) region of ribosomal DNA and the cytochrome C oxidase subunit 1 (CoxI) gene and was shown to be specific only to Saprolegnia genus. This LAMP method can detect as low as 10 fg of S. salmonis DNA while the qPCR method has a detection limit of 2 pg of S. salmonis DNA, indicating the superior sensitivity of LAMP compared to qPCR. When applied to detect the pathogen in water samples, both methods could detect the pathogen when only one zoospore of Saprolegnia was present. We propose LAMP as a quick (about 20–60 minutes) and sensitive molecular diagnostic tool for the detection of Saprolegnia spp. suitable for on-site applications.

Targeted Hsp70 fluorescence molecular endoscopy detects dysplasia in Barrett’s esophagus

Purpose The incidence of esophageal adenocarcinoma (EAC) has been increasing for decades without significant improvements in treatment. Barrett’s esophagus (BE) is best established risk factor for EAC, but current surveillance with random biopsies cannot predict progression to cancer in most BE patients due to the low sensitivity and specificity of high-definition white light endoscopy. Methods Here, we evaluated the membrane-bound highly specific Hsp70-specific contrast agent Tumor-Penetrating Peptide (Hsp70-TPP) in guided fluorescence molecular endoscopy biopsy. Results Hsp70 was significantly overexpressed as determined by IHC in dysplasia and EAC compared with non-dysplastic BE in patient samples (n = 12) and in high-grade dysplastic lesions in a transgenic (L2-IL1b) mouse model of BE. In time-lapse microscopy, Hsp70-TPP was rapidly taken up and internalized by human BE dysplastic patient–derived organoids. Flexible fluorescence endoscopy of the BE mouse model allowed a specific detection of Hsp70-TPP-Cy5.5 that corresponded closely with the degree of dysplasia but not BE. Ex vivo application of Hsp70-TPP-Cy5.5 to freshly resected whole human EAC specimens revealed a high (> 4) tumor-to-background ratio and a specific detection of previously undetected tumor infiltrations. Conclusion In summary, these findings suggest that Hsp70-targeted imaging using fluorescently labeled TPP peptide may improve tumor surveillance in BE patients.

SPECIFIC DETECTION OF SARS-COV-2 B.1.1.529 (OMICRON) VARIANT BY FOUR RT-qPCR DIFFERENTIAL ASSAYS

In this report, we describe four RT-qPCR assays that enable rapid identification of the newly emerging SARS-COV-2 Omicron (B.1.1.529) variant of concern. The assays target Omicron characteristic mutations in the nsp6 (Orf1a), spike and nucleocapsid genes. We demonstrate that the assays are straightforward to assemble and perform, are amendable for multiplexing, and may be used as a reliable first-line tool to identify B.1.1.529 suspected samples. Importantly, this is a preliminary development report. Further validation and optimization of the assays described herein will be published hereafter.