Siddiqi, I. | bioRxiv Science

bioRxiv2020

An Inexpensive RT-PCR Endpoint Diagnostic Assay for SARS-CoV-2 Using Nested PCR: Direct Assessment of Detection Efficiency of RT-qPCR Tests and Suitability for Surveillance

With a view to extending testing capabilities for the ongoing SARS-CoV-2 pandemic we have developed a test that lowers cost and does not require real time quantitative reverse transcription polymerase chain reaction (RT-qPCR). We developed a reverse transcription nested PCR endpoint assay (RT-nPCR) and showed that RT-nPCR has comparable performance to the standard RT-qPCR test. In the course of comparing the results of both tests, we found that the standard RT-qPCR test can have low detection efficiency (less than 50%) in a real testing scenario which may be only partly explained by low viral representation in many samples. This finding points to the importance of directly monitoring detection efficiency in test environments. We also suggest measures that would improve detection efficiency.

molecular biology↗

bioRxiv2019

The TARANI/ UBIQUITIN SPECIFIC PROTEASE 14 destabilizes the AUX/IAA transcriptional repressors and regulates auxin response in Arabidopsis thaliana

Auxin response is regulated by a group of AUX/IAA transcriptional inhibitors that suppress auxin signaling in the absence of the hormone. While the degradation of these proteins upon auxin signaling has been well studied, the molecular control of their rapid turn-over is not clearly understood. Here, we report that the TARANI/ UBIQUITIN PROTEASE 14 protein in Arabidopsis thaliana (Arabidopsis) is required for AUX/IAA degradation. The tni mutation was originally identified in a forward genetic screen to isolate mutants with altered leaf shape. Detailed phenotypic analysis revealed that tni displays pleiotropic phenotypic alterations that resemble auxin-related defects. The activity of auxin responsive reporters DR5::GUS, DR5::nYFP and IAA2::GUS was reduced in tni organs, implying that TNI is required for normal auxin response. Genetic interaction studies suggested that TNI acts along with TIR1, ARF7, AUX1 and PIN1 - molecules involved in auxin signaling or transport. A map-based cloning approach combined with next-generation sequencing identified TNI as UBIQUITIN SPECIFIC PROTEASE14 which is involved in ubiquitin recycling. In tni, the mutant primary transcript is spliced inefficiently, which is predicted to produce an aberrant protein product in addition to the normal protein, where a polypeptide corresponding to the 3rd intron in inserted in-frame within the Zn-finger domain of UBP14. The tni plants accumulated poly-ubiquitin chains and excess poly-ubiquitinated proteins due to reduced TNI activity. Improper ubiquitin recycling affected the degradation of DII:VENUS, IAA18:GUS and HS::AXR3-NT:GUS, resulting in their stabilization in the tni mutant. Thus, our study identified a function for TNI/UBP14 in regulating auxin response through ubiquitin recycling.

plant biology↗