A Sphingobium sp. strain isolated from radioactive solid waste management site (RSMS) completely degraded 7.98 g/L of tributyl phosphate (TBP) from TBP containing suspensions in 3 days. It also completely degraded 20 mM dibutyl phosphate (DBP) within 2 days. The strain tolerated high levels of TBP and showed excellent stability with respect to TBP degradation over several repeated subcultures. On solid minimal media or Luria Bertani media supplemented with TBP, the RSMS strain showed a clear zone of TBP degradation around the colony. Gas chromatography and spectrophotometry analyses identified DBP as the intermediate and butanol and phosphate as the products of TBP biodegradation. The RSMS strain utilized both TBP and DBP as the sole source of carbon and phosphorous for its growth. The butanol released was completely utilized by the strain as a carbon source thereby leaving no toxic residue in the medium. Degradation of TBP or DBP was found to be suppressed by high concentration of glucose which also inhibited TBP or DBP dependent growth. The results highlight the potential of Sphingobium sp. strain RSMS for bioremediation of TBP and for further molecular investigation.

Aim The current scenario of COVID‐19 pandemic has presented an almost insurmountable challenge even for the most sophisticated hospitals equipped with modern biomedical technology. There is an urgency to develop simple, fast and highly accurate methods for the rapid identification and isolation of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infected patients. To address the ongoing challenge, the present study offers a CLEVER assay ( C RISPR‐Cas integrated RT‐ L AMP E asy, V isual and E xtraction‐free R NA) which will allow RNA extraction‐free method to visually diagnose COVID‐19. RNA extraction is a major hurdle in preventing rapid and large‐scale screening of samples particularly in low‐resource regions because of the logistics and costs involved. Method and Result Herein, the visual SARS‐CoV‐2 detection method consists of RNA extraction‐free method directly utilizing the patient's nasopharyngeal and oropharyngeal samples for reverse transcription loop‐mediated isothermal amplification (RT‐LAMP). Additionally, the assay also utilizes the integration of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)‐Cas12‐based system using different guide RNAs of N, E and an internal control POP7 (human RNase P) genes along with visual detection via lateral flow readout‐based dip sticks with unaided eye (~100 min). Overall, the clinical sensitivity and specificity of the CLEVER assay were 89.6% and 100%, respectively. Conclusion Together, our CLEVER assay offers a point‐of‐care tool with no equipment dependency and minimum technical expertise requirement for COVID‐19 diagnosis. Significance and Impact of the Study To address the challenges associated with COVID‐19 diagnosis, we need a faster, direct and more versatile detection method for an efficient epidemiological management of the COVID‐19 outbreak. The present study involves developing a method for detection of SARS‐CoV‐2 in human body without RNA isolation step that can visually be detected with unaided eye. Taken together, our assay offers to overcome one major defect of the prior art, that is, RNA extraction step, which could limit the deployment of the previous assays in a testing site having limited lab infrastructure.

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A tri- and dibutyl phosphate (TBP/DBP) non-degrading spontaneous mutant, Sphingobium SS22, was derived from the Sphingobium sp. strain RSMS (wild type). Unlike the wild type strain, Sphingobium SS22 could not grow in a minimal medium supplemented with TBP or DBP as the sole source of carbon or phosphorous. Sphingobium SS22 also did not form any of the intermediates or end products of TBP or DBP degradation, namely DBP, butanol or inorganic phosphate. Proteomic analysis revealed the absence of three prominent proteins in Sphingobium SS22 as compared to wild type. These proteins were identified by MALDI mass spectrometry, and they showed similarities to phosphohydrolase- and exopolyphosphatase-like proteins from other bacteria, which belong to the class of phosphoesterases. Cellular proteins of Sphingobium SS22 showed none or negligible phosphodiesterase (PDE) and phosphomonoesterase (PME) activities at pH 7 and displayed approximately five- and approximately twofold less DBP and monobutyl phosphate (MBP) degradation activity, respectively, in comparison to the wild type strain. In-gel zymographic analysis revealed two PDE and PME activity bands in the wild type strain, one of which was absent in the Sphingobium SS22 mutant. The corresponding proteins from the wild type strain could degrade DBP and MBP. The results demonstrate the involvement of phosphoesterase enzymes in the TBP degradation pathway elucidated earlier.

Aims The RT‐PCR is the most popular confirmatory test for SARS‐CoV‐2. It is sensitive, but high instrumentation cost makes it difficult for use outside routine clinical setup. This has necessitated the development of alternative methods such as CRISPR‐based DETECTR method which uses lateral flow technology. Although accurate and sensitive, this method is limited by complex steps and recurrent cost of high‐quality lateral flow strips. The main goal of this study was to improve the Cas12a‐based SARS‐CoV‐2 DETECTR method and develop a portable and field‐deployable system to reduce the recurring consumable cost. Methods and results Specific regions of N and E genes from SARS‐CoV‐2 virus and human RNase P (internal control) were reverse transcribed (RT) and amplified by loop‐mediated isothermal amplification (LAMP). The amplified products were detected by a Cas12a‐based trans‐cleavage reaction that generated a fluorescent signal which could be easily visualized by naked eye. Detection of internal control, RNase P gene was improved and optimized by redesigning RT‐LAMP primers. A number of steps were reduced by combining the reagents related to the detection of Cas12a trans‐cleavage reaction into a single ready‐to‐use mix. A portable, cost‐effective battery‐operated instrument, CRISPR‐CUBE was developed to run the assay and visualize the outcome. The method and instrument were validated using both contrived and patient samples. Conclusions The simplified CRISPR‐based SARS‐CoV‐2 detection and instrument developed in this study, along with improved design for internal control detection allows for easier, more definitive viral detection requiring only reagents, consumables and the battery operable CRISPR‐CUBE. Significance and impact of study Significant improvement in Cas12 method, coupled with simple visualization of end point makes the method and instrument deployable at the point‐of‐care (POC) for SARS‐CoV‐2 detection, without any recurrent cost for the lateral flow strips which is used in other POC methods.

ObjectiveTo identify monkeypox-specific sequence and distinguish it from related orthopoxviruses followed by development of a CRISPR-Cas12a-based, specific and sensitive detection of monkeypox virus.MethodsA common detection mixture (CDM) was constituted comprising of CRISPR RNAs (crRNAs) for general orthopoxviruses and monkeypox virus-specific targets along with LbCas12a and fluorescent reporter. Recombinase polymerase amplification (RPA) of target loci was carried out followed by detection using the CRISPR-Cas12 CDM complex. Fluorescence-based read out can be monitored by a fluorescent reader and alternatively can also be visualized under a blue light illuminator by naked eye.ResultsMonkeypox-specific conserved sequences were identified inpolAgene which differ by a single nucleotide polymorphism (SNP) from all the viruses present in genus Orthopoxvirus. Our Cas12a-based assay was capable of specifically distinguishing monkeypox virus from other related orthopoxviruses with an LOD of 60 copies in 1 hour after the initiation of the reaction.ConclusionOur assay exhibits sensitive and specific detection of monkeypox virus which can prove to be of practical value for surveillance in areas infected with multiple orthopoxviruses, especially in hotspots of monkeypox virus infections.

Sphingobium sp. strain RSMS was described earlier as an efficient degrader of tributyl phosphate, an organic pollutant. This report describes the generation and annotation of the genome sequence of Sphingobium sp. strain RSMS, which will facilitate future studies to identify genetic elements responsible for the degradation of tributyl phosphate.

Mpox virus, a member of genus Orthopoxvirus, causes rash and flu‐like symptoms in humans. In the recent global outbreak, it was reported from several geographical areas that have not historically reported mpox. Point of care, sensitive and specific mpox diagnostic assays are critical in checking the spread of the disease. We have developed a clustered regularly interspaced short palindromic repeats associated Cas12a nuclease‐based assay for detecting mpox virus. Mpox specific conserved sequences were identified in polA (E9L) gene which differ by a single nucleotide polymorphism (SNP) from all the viruses present in the genus Orthopoxvirus. This SNP was exploited in our assay to specifically distinguish mpox virus from other related orthopox viruses with a limit of detection of 1 copy/μl in 30 min. The assay exhibits a sensitive and specific detection of mpox virus which can prove to be of practical value for its surveillance in areas infected with multiple orthopox viruses, especially in hotspots of mpox virus infections.