Reverse Transcriptase Loop-mediated Amplification Research Articles

Capillary-based reverse transcriptase loop-mediated isothermal amplification for cost-effective and rapid point-of-care COVID-19 testing

As the SARS-CoV-2 pandemic continues to spread, the necessity for rapid, easy diagnostic capabilities could never have been more crucial. With this aim in mind, we have developed a cost-effective and time-saving testing methodology/strategy that implements a sensitive reverse transcriptase loop-mediated amplification (RT-LAMP) assay within narrow, commercially available and cheap, glass capillaries for detection of the SARS-CoV-2 viral RNA. The methodology is compatible with widely used laboratory-based molecular testing protocols and currently available infrastructure. It employs a simple rapid extraction protocol that lyses the virus, releasing sufficient genetic material for amplification. This extracted viral RNA is then amplified using a SARS-CoV-2 RT-LAMP kit, at a constant temperature and the resulting amplified product produces a colour change which can be visually interpreted. This testing protocol, in conjunction with the RT-LAMP assay, has a sensitivity of ∼100 viral copies per reaction of a sample and provides results in a little over 30 min. As the assay is carried out in a water bath, commonly available within most testing laboratories, it eliminates the need for specialised instruments and associated skills. In addition, our testing pathway requires a significantly reduced quantity of reagents per test while providing comparable sensitivity and specificity to the RT-LAMP kit used in this study. While the conventional technique requires 25 μl of reagent, our test only utilises less than half the quantity (10 μl). Thus, with its minimalistic approach, this capillary-based assay could be a promising alternative to the conventional testing, owing to the fact that it can be performed in resource-limited settings, using readily available apparatus, and has the potential of increasing the overall testing capacity, while also reducing the burden on supply chains for mass testing.

A Novel Approach to the Bioluminescent Detection of the SARS-CoV-2 ORF1ab Gene by Coupling Isothermal RNA Reverse Transcription Amplification with a Digital PCR Approach.

The COVID-19 pandemic caused by the SARS-CoV-2 virus, which first emerged in December 2019, represents an ongoing global public health emergency. Here, we developed an improved and highly sensitive approach to SARS-CoV-2 detection via coupling bioluminescence in real-time (BART) and reverse-transcriptase loop-mediated amplification (RT-LAMP) protocols (RT-LAMP-BART) and was also compatible with a digital LAMP system (Rainsuit), which did not allow for real-time quantification but did, nonetheless, facilitate absolute quantification with a comparable detection limit of 104 copies/mL. Through improving RNA availability in samples to ensure the target RNA present in reaction, we additionally developed a simulated digital RT-LAMP approach using this same principle to enlarge the overall reaction volume and to achieve real-time detection with a limit of detection of 10 copies/mL, and with further improvements in the overall dynamic range of this assay system being achieved through additional optimization.

Optimization and clinical validation of dual-target RT-LAMP for SARS-CoV-2

A novel reverse-transcriptase loop mediated amplification (RT-LAMP) method targeting genes encoding the Spike (S) protein and RNA-dependent RNA polymerase (RdRP) of SARS-CoV-2 has been developed. The LAMP assay achieves a comparable limit of detection (25–50 copies per reaction) to commonly used RT-PCR protocols using clinical samples quantified by digital droplet PCR. Precision, cross-reactivity, inclusivity, and limit of detection studies were performed according to regulatory standards. Clinical validation of dual-target RT-LAMP (S and RdRP gene) achieved a PPA of 98.48 % (95 % CI 91.84%–99.96%) and NPA 100.00 % (95 % CI 93.84%–100.00%) based on the E gene and N2 gene reference RT-PCR methods. The method has implications for development of point of care technology using isothermal amplification.

May axillary dissection be avoided after micro metastases in sentinel lymph node analyzed by OSNA method?

18 Background: The study of the sentinel node biopsy is a common method to assess axillary involvement before surgical resection. The OSNA assay based on reverse transcriptase loop mediated amplification (RT-LAMP) and quantification of cytokeratin 19 (CK19) mRNA allows a rapid intra-operative detection of the metastases in the sentinel lymph node in breast cancer patients. Pathologists considered micro metastases when the size of the node involvement was between 0.2 and 2 mm and the OSNA method assess micro metastases when the copy number of CK19 is between 250 and 5000. Methods: From a total of 341 patients in which sentinel node had been analyzed, we studied a series of 32 patients whose sentinel nodes had micro metastases assessed by OSNA and who underwent subsequent axillary dissection. Median breast tumour size was 1.6 cm, 10 patients (31%) had less than 1 cm in size and only 6 patients (19%) had >2 cm. 78% of patients shown positive oestrogen receptor and 14 patients (44%) had low Ki67 (<14%). The CK19 mRNA was quantified in all patients (32) resulting in a median value of 540 (210-4800). Results: The median value of the nodes removed by axillary dissection was 14 (9-19). We found tumor invasion in none of them by histopathological conventional study. Conclusions: Since we have found no axillary involvement when the sentinel node have micro metastases analyzed by the OSNA method it seems that quantitative measure of CK19 by OSNA may be a useful procedure in order to avoid axillary dissection, although it would be necessary to perform larger studies to demonstrate this effect.