An expanded RT-PCR melting temperature coding assay to rapidly identify all known SARS-CoV-2 variants and sub-variants of concern.

The continued emergence of vaccine-resistant SARS-CoV-2 variants of concern (VOC) requires specific identification of each VOC as it arises. Here, we report an expanded version of our previously described sloppy molecular beacon (SMB) melting temperature (Tm) signature-based assay for VOCs, now modified to include detection of Delta (B.1.617.2) and Omicron (B.1.1.529) sub-variants. The SMB-VOC assay targets the signature codons 501, 484 and 452 in the SARS-CoV-2 spike protein which we show can specifically detect and differentiate all known VOCs including the Omicron subvariants (BA.1, BA.2, BA.2.12.1, BA.4/BA.5). The limit of detection (LOD) of the assay was 20, 22 and 36 genomic equivalents (GE) per reaction with the Delta, Omicron BA.1 and BA.2 respectively. Clinical validation of the 3-codon assay in the LC480 instrument showed the assay detected 94% (81/86) of the specimens as WT or VOCs and 6% (5/86) of the tests producing indeterminate results compared to sequencing. Sanger sequencing also failed for four samples. None of the specimens were incorrectly identified as WT or as a different VOC by our assay. Thus, excluding specimens with indeterminant results, the assay was 100% sensitive and 100% specific compared to Sanger sequencing for variant identification. This new assay concept can be easily expanded to add newer variants and can serve as a robust diagnostic tool for selecting appropriate monoclonal antibody therapy and rapid VOC surveillance.

A bacterial signature-based method for the identification of seven forensically relevant human body fluids.

Detection and identification of body fluids plays a crucial role in criminal investigation, as it provides information on the source of the DNA as well as corroborative evidence regarding the crime committed, scene, and/or association with persons of interest. Historically, forensic serological methods have been chemical, immunological, catalytic, spectroscopic, and/or microscopic in nature. However, most of these methods are presumptive, with few robust confirmatory exceptions. In recent years several new molecular methods (mRNA, miRNA, DNA methylation, etc.) have been proposed; although promising, these methods require high quality human DNA or RNA. Additional steps are required in RNA based methods. Additionally, RNA based methods cannot be used for old cases where only DNA extracts remain to sample from. In this study, a novel non-human DNA (microbiome) based method was developed for the identification of the majority of forensically relevant human biological samples. Eight hundred and twelve (n = 812) biological samples (semen, vaginal fluid, menstrual blood, saliva, feces, urine, and blood) were collected and preserved using methods commonly used in forensic laboratories for evidence collection. Variable region four (V4) of 16 S ribosomal DNA (16 S rDNA) was amplified using a dual-indexing strategy and then sequenced on the MiSeq FGx sequencing platform using the MiSeq Reagent Kit v2 (500 cycles) and following the manufacturer's protocol. Machine learning prediction models were used to assess the classification accuracy of the newly developed method. As there was no significant difference in bacterial communities between vaginal fluid, menstrual blood, and female urine, these were combined as female intimate samples. Except in urine, the bacterial structures associated with male and female body fluid samples were not significantly different from one another. The newly developed method accurately identified human body fluid samples with an overall accuracy of more than 88%. This newly developed bacterial signature-based method is fast (no additional steps are needed as the same DNA can be used for both body fluid identification and STR typing), efficient (consume less sample as a single test can identify all major body fluids), sensitive (needs only 5 pg of bacterial DNA), accurate, and can be easily added into a forensic high throughput sequencing (HTS) panel.

Player-avatar interactions in habitual and problematic gaming: A qualitative investigation.

BACKGROUND AND AIMS: Previous studies have reported that stronger avatar identification and negative self-concept are associated with gaming disorder (GD). This study aimed to examine the value and significance of avatars based on firsthand accounts from regular and problematic gamers, and to identify any potential links between avatar-related experiences and excessive gaming. METHODS: An online survey of 993 adult gamers yielded 3,972 text responses. Qualitative analysis of 59,059 words extracted 10 categories of avatar-related perspectives. RESULTS: Some problem and non-problem gamers employed sentimental language (e.g., 'dear friend', 'like a child', 'part of my soul') to refer to their avatar. However, most participants perceived avatars as a means of achieving in-game goals and enabling greater interactivity (e.g., socializing). When asked to reflect on hypothetically losing their avatar, participants generally anticipated feeling temporary frustration or annoyance due to lost time and effort invested into the avatar. Although some participants reported that their avatar 'mattered', avatars were often considered as superficial ('just pixels') and peripheral to the primary reinforcement of achieving in-game rewards and objectives. Some broader psychological and identity issues such as gender dysphoria, rather than 'addiction', were cited as motivating persistent avatar-related interactions and attachment. DISCUSSION AND CONCLUSIONS: Participants reported diverse views on the psychological value and function of avatars, but the relationship between avatars and problematic gaming or GD was largely unclear or inconsistent, and refuted by some participants. Future research with clinical samples may lead to a better understanding of player-avatar processes, including whether avatar-stimuli facilitate the development of maladaptive gaming habits, particularly among psychologically vulnerable players. Future investigations should be mindful of 'overpathologizing' avatar-related phenomena and recognize their important role in socializing, storytelling, and creative expression among gamers.

A Simple Reverse Transcriptase PCR Melting-Temperature Assay To Rapidly Screen for Widely Circulating SARS-CoV-2 Variants.

The increased transmission of SARS-CoV-2 variants of concern (VOC), which originated in the United Kingdom (B.1.1.7/alpha), South Africa (B1.351/beta), Brazil (P.1/gamma), the United States (B.1.427/429 or epsilon), and India (B.1.617.2/delta), requires a vigorous public health response, including real-time strain surveillance on a global scale. Although genome sequencing is the gold standard for identifying these VOCs, it is time-consuming and expensive. Here, we describe a simple, rapid, and high-throughput reverse transcriptase PCR (RT-PCR) melting-temperature (Tm) screening assay that identifies the first three major VOCs. RT-PCR primers and four sloppy molecular beacon (SMB) probes were designed to amplify and detect the SARS-CoV-2 N501Y (A23063T) and E484K (G23012A) mutations and their corresponding wild-type sequences. After RT-PCR, the VOCs were identified by a characteristic Tm of each SMB. Assay optimization and testing was performed with RNA from SARS-CoV-2 USA WA1/2020 (wild type [WT]), B.1.1.7, and B.1.351 variant strains. The assay was then validated using clinical samples. The limit of detection for both the WT and variants was 4 and 10 genomic copies/reaction for the 501- and 484-codon assays, respectively. The assay was 100% sensitive and 100% specific for identifying the N501Y and E484K mutations in cultured virus and in clinical samples, as confirmed by Sanger sequencing. We have developed an RT-PCR melt screening test for the major VOCs that can be used to rapidly screen large numbers of patient samples, providing an early warning for the emergence of these variants and a simple way to track their spread.

A Simple RT-PCR Melting temperature Assay to Rapidly Screen for Widely Circulating SARS-CoV-2 Variants.

BACKGROUND: The increased transmission of SARS-CoV-2 variants of concern (VOC) which originated in the United Kingdom (B.1.1.7), South Africa (B1.351), Brazil (P.1) and in United States (B.1.427/429) requires a vigorous public health response, including real time strain surveillance on a global scale. Although genome sequencing is the gold standard for identifying these VOCs, it is time consuming and expensive. Here, we describe a simple, rapid and high-throughput reverse-transcriptase PCR (RT-PCR) melting temperature (Tm) screening assay that identifies these three major VOCs. METHODS: RT-PCR primers and four sloppy molecular beacon (SMB) probes were designed to amplify and detect the SARS-CoV-2 N501Y (A23063T) and E484K (G23012A) mutations and their corresponding wild type sequences. After RT-PCR, the VOCs were identified by a characteristic Tm of each SMB. Assay optimization and testing was performed with RNA from SARS-CoV-2 USA WA1/2020 (WT), a B.1.17 and a B.1.351 variant strains. The assay was then validated using clinical samples. RESULTS: The limit of detection (LOD) for both the WT and variants was 4 and 10 genomic copies/reaction for the 501 and 484 codon assays, respectively. The assay was 100% sensitive and 100% specific for identifying the N501Y and E484K mutations in cultured virus and in clinical samples as confirmed by Sanger sequencing. CONCLUSION: We have developed an RT-PCR melt screening test for the three major VOCs which can be used to rapidly screen large numbers of patient samples providing an early warning for the emergence of these variants and a simple way to track their spread.

Avatar identification and problematic gaming: The role of self-concept clarity.

Some video-gaming activities feature customizable avatars that enable users to fulfil self-identity needs. Research evidence (e.g., fMRI and survey studies) has suggested that poorer self-concept and stronger avatar identification are associated with problematic gaming. Player-avatar relationships have thus been proposed to require attention in gaming disorder assessment and interventions. To examine the interplay of player-avatar interactions in problematic gaming, this study investigated whether avatar identification differed according to avatar characteristics and game types, and whether the association between avatar identification and problem gaming was mediated by self-concept clarity. A total of 993 adult respondents completed an online survey that assessed problematic gaming, avatar identification, and self-concept clarity. The results indicated that avatar identification scores were generally unrelated to avatar characteristics (e.g., human resemblance, degree of customizability, and in-game perspective). Avatar identification was significantly positively related to problematic gaming and significantly negatively related to self-concept clarity. There was a significant indirect relationship between avatar identification on problem gaming mediated through self-concept clarity. These findings suggest that poorer self-concept clarity may be one mechanism by which avatar identification affects problem gaming. Future research with clinical samples may help to gain a better understanding of avatar-related processes and psychological vulnerabilities related to problematic gaming.

Avatar- and self-related processes and problematic gaming: A systematic review.

The concept of self has become increasingly relevant to understanding the psychological mechanisms of problematic online gaming. Many gaming activities feature in-game avatars that enable the adoption of, and experimentation with, different roles and identities. Avatars enable players to compensate for perceived deficiencies in their real-world self (e.g., lack of physical strength, beauty, or social status). Currently, how avatar- and self-related processes may develop and maintain problematic gaming, including gaming disorder (GD), is unclear. This systematic review examined 18 quantitative studies of avatar- and self-related concepts and problematic gaming, including 13 survey-based and 5 neuroimaging studies. Despite variability in the conceptualization and measurement of avatar/self-related concepts, survey-based studies have consistently reported that negative self-concept, avatar identification, and large self-avatar discrepancies are significantly associated with problematic gaming. Poor self-concept appears to be a risk factor for GD, particularly for games that facilitate role-playing and identity formation. Further research and clinical evidence are needed to explain how avatar- and self-related processes may relate to the addictive mechanisms of GD (e.g., cognitive distortions, reward-seeking, inhibitory control, self-regulation systems), amid calls for problem gaming-related assessment and interventions to incorporate a focus on avatar identification.

microRNA Detection in Blood, Urine, Semen, and Saliva Stains After Compromising Treatments.

Evaluation of microRNA (miRNA) expression as a potential method for forensic body fluid identification has been the subject of investigation over the past several years. Because of their size and encapsulation within proteins and lipids, miRNAs are inherently less susceptible to degradation than other RNAs. In this work, blood, urine, semen, and saliva were exposed to environmental and chemical conditions mimicking sample compromise at the crime scene. For many treated samples, including 100% of blood samples, miRNAs remained detectable, comparable to the untreated control. Sample degradation varied by body fluid and treatment, with blood remarkably resistant, while semen and saliva are more susceptible to environmental insult. Body fluid identification using relative miRNA expression of blood and semen of the exposed samples was 100% and 94%, respectively. Given the overall robust results herein, the case is strengthened for the use of miRNAs as a molecular method for body fluid identification.

An accurate bacterial DNA quantification assay for HTS library preparation of human biological samples.

Sequencing and classification of microbial taxa within forensically relevant biological fluids has the potential for applications in the forensic science and biomedical fields. The quantity of bacterial DNA from human samples is currently estimated based on quantity of total DNA isolated. This method can miscalculate bacterial DNA quantity due to the mixed nature of the sample, and consequently library preparation is often unreliable. We developed an assay that can accurately and specifically quantify bacterial DNA within a mixed sample for reliable 16S ribosomal DNA (16S rDNA) library preparation and high throughput sequencing (HTS). A qPCR method was optimized using universal 16S rDNA primers, and a commercially available bacterial community DNA standard was used to develop a precise standard curve. Following qPCR optimization, 16S rDNA libraries from saliva, vaginal and menstrual secretions, urine, and fecal matter were amplified and evaluated at various DNA concentrations; successful HTS data were generated with as low as 20 pg of bacterial DNA. Changes in bacterial DNA quantity did not impact observed relative abundances of major bacterial taxa, but relative abundance changes of minor taxa were observed. Accurate quantification of microbial DNA resulted in consistent, successful library preparations for HTS analysis.