Performance Evaluation of the SAMBA II SARS-CoV-2 Test for Point-of-Care Detection of SARS-CoV-2.

Nucleic acid amplification for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in respiratory samples is the standard method for diagnosis. The majority of this testing is centralized and therefore has turnaround times of several days. Point-of-care (POC) testing with rapid turnaround times would allow more effective triage in settings where patient management and infection control decisions need to be made rapidly. The inclusivity and specificity of the Simple AMplification-Based Assay (SAMBA) II SARS-CoV-2 test were determined by both in silico analyses of the primers and probes and wet testing. The SAMBA II SARS-CoV-2 test was evaluated for performance characteristics. Clinical performance was evaluated in residual combined throat/nose swabs and compared to that of the Public Health England real-time PCR assay targeting the RdRp gene. The SAMBA II SARS-CoV-2 test has an analytical sensitivity of 250 copies/ml for detecting two regions of the genome (open reading frame 1ab [ORF1ab] and nucleocapsid protein [N]). The clinical performance was evaluated in 172 residual combined nose/throat swabs provided by the Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Cambridge (CMPHL), which showed an estimated positive percent agreement of 98.9% (95% confidence interval [CI], 93.83 to 99.97) and negative percent agreement of 96.4% (95% CI, 89.92 to 99.26) compared to testing by the CMPHL. The data show that the SAMBA II SARS-CoV-2 test performs equivalently to the centralized testing methods, but with a shorter turnaround time of 86 to 101 min. Point-of-care tests such as SAMBA should enable rapid patient management and effective implementation of infection control measures.

Contribution of nonconsensus base pairs within ArsR binding sequences toward ArsR-DNA binding and arsenic-mediated transcriptional induction.

BACKGROUND: A transcriptional reporter is the key component in bacterial biosensors which are employed to monitor the induction or repression of a reporter gene corresponding to environmental change. Interaction of a transcription factor with its consensus sequence generated by using a position weight matrix (PWM) model is crucial for its sensitivity of the reporter. However, recent studies suggest that PWM model based on independent contribution of individual consensus base pairs to protein interaction is often insufficient to explain complex regulation, such as the effect of nonconsensus sequences on the protein-DNA binding affinity. In the present study, we employed a simpler prokaryotic arsenic repressor (ArsR) regulation system to access the protein-DNA recognition. Contribution of nonconsensus base pairs within ArsR binding sequences toward ArsR-DNA binding and arsenic-mediated transcriptional induction was studied. RESULTS: We constructed a series of arsenic responsive reporters, each comprising two copies of the ArsR binding sequences from different resources. We found that high arsenic-mediated induction specifically requires the binding sequence from Escherichia coli to be placed at the first binding sequence; however, no such preference was observed for the second binding sequence, which could be from Acidithiobacillus ferrooxidans, plasmid R773, Synechococcus, or a core binding sequence of arsR. By creating a series of reporters differed at the nonconsensus base pairs of the second binding sequence, we observed that some constructs bound weakly while others strongly to ArsR. Most interestingly, although a number of these reporters showed similar binding affinity to ArsR, their arsenic-dependent induction differed significantly. CONCLUSIONS: The results indicated that nonconsensus base pairs could have profound influence on protein binding and may also modulate post-binding function. These findings provide new insights into the complex regulation of gene expression and facilitate the development of transcriptional reporter-based biosensors.

Copy number of ArsR reporter plasmid determines its arsenite response and metal specificity.

The key component in bacteria-based biosensors is a transcriptional reporter employed to monitor induction or repression of a reporter gene corresponding to environmental change. In this study, we made a series of reporters in order to achieve highly sensitive detection of arsenite. From these reporters, two biosensors were developed by transformation of Escherichia coli DH5α with pLHPars9 and pLLPars9, consisting of either a high or low copy number plasmid, along with common elements of ArsR-luciferase fusion and addition of two binding sequences, one each from E. coli and Acidithiobacillus ferrooxidans chromosome, in front of the R773 ArsR operon. Both of them were highly sensitive to arsenite, with a low detection limit of 0.04 µM arsenite (~ 5 µg/L). They showed a wide dynamic range of detection up to 50 µM using high copy number pLHPars9 and 100 µM using low copy number pLLPars9. Significantly, they differ in metal specificity, pLLPars9 more specific to arsenite, while pLHPars9 to both arsenite and antimonite. The only difference between pLHPars9 and pLLPars9 is their copy numbers of plasmid and corresponding ratios of ArsR to its binding promoter/operator sequence. Therefore, we propose a working model in which DNA bound-ArsR is different from its free form in metal specificity.

An in vivo approach to isolating allosteric pathways using hybrid multimeric proteins.

Hybrid tetramers of Escherichia coli phosphofructokinase (EC 2.7.1.11; EcPFK) have been used to dissect the complicated allosteric interactions within the native tetramer. The method used previously to generate hybrids in vitro involves dissociation of the parent proteins with KSCN followed by re-association as KSCN is removed via dialysis. However, this procedure is time consuming and is plagued with low hybrid yields. Consequently, we have attempted to produce hybrids more quickly and with potentially higher yields in vivo by co-expressing the parental EcPFK protein in E. coli. Wild-type EcPFK gene was cloned into pALTER-Ex2 and pALTER-1, respectively. Site-directed mutagenesis was performed to make mutant EcPFK gene in pALTER-1. Since each vector has a different origin of replication and antibiotic selection marker, we were able to co-transform both plasmids to competent E. coli cells. Following an affinity purification column, anion-exchange chromatography was used to separate the five hybrid species (4:0, 3:1, 2:2, 1:3, 0:4). While all five hybrid species were obtained, the amount 1:3 and 0:4 hybrids were very small. By changing the expression vector for the mutant EcPFK protein from pALTER-1 to pALTER-Ex1 and the charge-tag mutations from K2E/K3E to K90E/K91E, the yield of 1:3 hybrid was substantially increased. The in vivo method does increase the yield of the hybrids produced while decreasing the time required for their isolation.

[Cloning of flocculent gene and expression in industrial yeast strain].

The partial genomic library of Saccharomyce cerevisiae FL189 possessing strong flocculation ability was constructed using Yeast-E. coli shuttle plasmid YCp50 as vector. Recombinant plasmid containing flocculation gene was obtained by screening the growth of transformants on the selective medium and measurement flocculation, designated as pCF1.pCF1 was introduced into industrial yeast strain PJ208-5-15. Six transformants PJ208-5-15-1(pCF1)-PJ208-5-15-6(pCF1) possessing strong flocculation ability were obtained. The results of Southern blot and restriction endonuclease analysis showed that the insert is about 4.3 kb and could hybridize with the probe (2.6 kb Eco RV fragment of FLO1). Flocculation ability assay indicated that the transformants possess strong flocculation ability. Hence, the gene controlling flocculation phenotype exists in the cloned DNA fragment. The restriction endonuclease analysis and the sequence analysis of the insert DNA fragment are in progress.