VaNGuard assay protocol for SARS-CoV-2 detection

This protocol presents the Variant Nucleotide Guard (VaNGuard) assay, which is robust towards viral mutations and can be performed on purified RNA or directly on nasopharyngeal (NP) swab samples. The procedure typically comprises three parts, namely sample preparation, RT-LAMP reaction, and Cas12a-based detection via fluorescence or lateral flow assay. Sample preparation from NP swabs involves Proteinase K digestion followed by heat inactivation. Purified RNA or digested NP swab samples are then added as templates into RT-LAMP reactions and incubated at 65ºC for 22 minutes. Next, enAsCas12a and ssDNA-probes are added and the reactions are incubated at 60ºC for another 5 minutes. End-point fluorescence can be detected by a plate reader or a real-time PCR machine. Alternatively, a lateral flow strip can be inserted into each reaction tube for equipment-free read-out. The VaNGuard assay is a rapid and convenient point-of-care test for SARS-CoV-2 and is applicable to resource poor settings.

Colorimetric RT-LAMP SARS-CoV-2 diagnostic sensitivity relies on color interpretation and viral load

The use of RT-LAMP (reverse transcriptase—loop mediated isothermal amplification) has been considered as a promising point-of-care method to diagnose COVID-19. In this manuscript we show that the RT-LAMP reaction has a sensitivity of only 200 RNA virus copies, with a color change from pink to yellow occurring in 100% of the 62 clinical samples tested positive by RT-qPCR. We also demonstrated that this reaction is 100% specific for SARS-CoV-2 after testing 57 clinical samples infected with dozens of different respiratory viruses and 74 individuals without any viral infection. Although the majority of manuscripts recently published using this technique describe only the presence of two-color states (pink = negative and yellow = positive), we verified by naked-eye and absorbance measurements that there is an evident third color cluster (orange), in general related to positive samples with low viral loads, but which cannot be defined as positive or negative by the naked eye. Orange colors should be repeated or tested by RT-qPCR to avoid a false diagnostic. RT-LAMP is therefore very reliable for samples with a RT-qPCR Ct < 30 being as sensitive and specific as a RT-qPCR test. All reactions were performed in 30 min at 65 °C. The use of reaction time longer than 30 min is also not recommended since nonspecific amplifications may cause false positives.

Role of Phytopathogenic Fungi in Forest Plant Breeding–Development of DNA-Based Quick Tests for Quality Assurance in Forest Plant Production

The production of healthy seed and plant material is a fundamental prerequisite for the establishment of ecologically stable and economically productive forest stands. As in the past, forest plant production is nevertheless threatened by harmful biotic factors, with new, invasive species playing an increasingly significant role as a result of climate change and globalization. DNA-based methods have significantly accelerated the detection of plant pathogens, but are still time-consuming, costly, and require extensive equipment. Loop-mediated isothermal amplification (LAMP) is an efficient and cost-effective alternative to conventional polymerase chain reaction (PCR). The LAMP reaction is performed as a one-step assay at constant temperature and can be evaluated visually. The ongoing project “TreeLAMP” has led to the establishment of an LAMP assay for Rhabdocline pseudotsugae, one of the most important needle pathogens of Douglas fir. To date, 32 sets of LAMP primers have been derived from the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) and tested. The results show clear differences between the primer sets both in terms of reaction rate and concentration of amplified products. Following extensive work targeting the optimization of the LAMP reaction, a method is now available that enables the reliable detection of R. pseudotsugae at a constant temperature (65 °C) and after a reaction time of 1.5 h. The detection limit is currently 0.02 pg/µL of R. pseudotsugae DNA. The current focus of the project is the optimization of DNA extraction. In addition to conventional DNA kits, methods that are specially adapted to the detection procedure are also under investigation. These will allow DNA extraction to occur faster and without any great technical effort.

An easy, reliable and rapid SARS-CoV2 RT-LAMP based test for Point-of-Care and diagnostic lab

This study presents and evaluates a rapid and all-in-one SARS-CoV-2 RT-LAMP based molecular detection system, including RNA extraction or not, for point-of-care or massive testing of naso-pharyngeal swabs. The point-of-care format uses LoopX©, a small portative device ensuring optimal LAMP reaction and automated reading with 95.2% and 95.5% sensitivity and specificity respectively. This system might also be useful for testing other sample types such as saliva.

Quantification of colorimetric isothermal amplification on the smartphone and its open-source app for point-of-care pathogen detection

The increasing risk of infectious pathogens, especially in the under-developed countries, is demanding the development of point-of-care (POC) nucleic acid testing in the low-resource setting conditions. Here, we describe a methodology for colorimetric quantitative analysis of nucleic acid using an easy-to-build smartphone-based platform, offering low-cost, portability, simplicity in operation, and user-friendliness. The whole system consists of a hand-held box equipped with a smartphone, a film heater, a white LED, a loop-mediated isothermal amplification (LAMP) chip, and a DC converter, and all the processes were powered by a portable battery of 5 V. Upon the amplification of the target gene by an Eriochrome Black T-mediated LAMP reaction, the color of the LAMP reaction was changed from violet to blue that was real-time recorded by a smartphone camera. To keep track of the progress of the color change, we developed a novel mobile app in which a hue value was accepted as an indicator for color transition and for determining the threshold time of the amplification reaction. A calibration curve could be generated by plotting the logarithm of the known concentration of the DNA templates versus the threshold time, and it can be used to predict the copy number of nucleic acids in the test samples. Thus, the proposed mobile platform can inform us of not only qualitative but also quantitative results of the pathogens. We believe that this advanced colorimetric approach and the mobile app can expand the potentials of the smartphone for the future POCT system in the bio-diagnostic fields.