Biology subjects

Kambakam, S.

Publications and source records attributed to Kambakam, S..

3 recordsLinked to original sources

bioRxiv2026

Confirmation of gamma irradiation mediated inactivation of a Vero adapted African swine fever virus Lisbon 60 strain for molecular assays

African swine fever virus (ASFV), the etiologic agent of African Swine Fever (ASF), is a high-consequence pathogen requiring experiments to be conducted in containment in non-endemic countries, thereby restricting diagnostic development, the creation of reference standards, and proficiency testing (PT). Safe and reliable inactivation methods are essential to expand diagnostic capacity while preserving nucleic acid integrity for molecular assays in unaffected countries. This study employed gamma irradiation to achieve complete inactivation of ASFV without compromising downstream molecular detection, as gamma irradiation offers deep penetration and uniform dose delivery. ASFV-cell culture supernatants were subjected to gamma irradiation doses ranging from 2 to 50 kGy. Viral replication was evaluated using TCID{square}{square} and serial passages, revealing a consistent dose{square}dependent reduction in infectivity across increasing irradiation dose levels and a complete loss of ASFV infectivity at 30 and 50 kGy. Molecular detection remained unaffected at all of the tested doses as confirmed by qPCR Ct values and sequence identity of the p72 gene. Whole genome sequencing demonstrated >99% genome coverage and consistent read depth profiles across irradiated and non-irradiated samples, indicating preservation of genomic integrity at all tested doses. These findings demonstrate that gamma irradiation at 50 kGy fully inactivates ASFV-cell supernatants while maintaining nucleic acid quality suitable for molecular diagnostics. The resulting inactivated material meets quality assurance requirements for molecular reference standards and PT panels and can be safely distributed to laboratories outside high containment facilities, supporting broader diagnostic readiness and harmonization of ASFV testing.

microbiology↗

bioRxiv2024

Rapid identification of African swine fever virus in diagnostic samples using CRISPR-Cas

African Swine Fever Virus (ASFV) is a high consequence, highly transmissible pathogen affecting swine causing African Swine Fever (ASF), a devastating disease, with high mortality rates in naive populations. Due to the likelihood of significant economic impacts associated with an ASF outbreak, considerable resources have been allocated in the United States (U.S.) to safeguard the swine industry against this threat. Ongoing outbreaks of ASF in the Dominican Republic and Haiti further threaten U.S. swine due to their proximity and involvement in movement to and from North America. While surveillance programs are ongoing, there are limited point-of-care (POC) tests available during outbreaks that maintain the sensitivity and specificity standards of laboratory testing (e.g., qPCR). However, the recently developed CRISPR-Cas testing systems may provide comparable high-quality results. In a CRISPR-based diagnostic assay, CRISPR effectors can be programmed with CRISPR-RNA (crRNA) to target specific DNA or RNA. Upon target binding, the Cas enzyme undergoes collateral cleavage of nearby fluorescently quenched reporter molecules (ssDNA or ssRNA), which can be detected under blue light or a fluorescence microplate reader. Furthermore, this tool is rapid, simple, cost-effective and can be performed with inexpensive equipment. For these reasons, we sought to develop a low-cost visual detection method for ASFV by employing the recombinase polymerase amplification (RPA)-dependent CRISPR-Cas12a technique that can be utilized in the field as a point-of-care-assay. Our CRISPR-Cas12a assay demonstrated comparable sensitivity and specificity to qPCR, both visually and when quantified using a fluorescent reader. In whole blood samples from ASFV-suspect or ASFV-negative cases, the CRISPR assay achieved a sensitivity of 98.3% (102 DNA copies) and a specificity of 100%. Finally, an assessment of the reaction time constraints indicated that results can be visualized in as little as seven minutes with a peak fluorescence at 40 min (RPA and CRISPR steps). The results of this feasibility assay validation allow for the rapid development of sensitive and specific POC tests that may be used for outbreak response in the future.

microbiology↗

bioRxiv2021

Zebrafish Cre/lox regulated UFlip alleles generated by CRISPR/Cas targeted integration provide cell-type specific conditional gene inactivation

The ability to regulate gene activity spatially and temporally is essential to investigate cell type specific gene function during development and in postembryonic processes and disease models. The Cre/lox system has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish, but simple and efficient methods for isolation of stable, Cre/lox regulated alleles are lacking. Here we applied our GeneWeld CRISPR/Cas9 short homology-directed targeted integration strategy to generate floxed conditional alleles that provide robust gene knockdown and strong loss of function phenotypes. A universal targeting vector, UFlip, with sites for cloning short 24-48 bp homology arms flanking a floxed mRFP gene trap plus secondary reporter cassette, was integrated into an intron in hdac1, rbbp4, and rb1. Active, gene off orientation hdac1-UFlip-Off and rb1-UFlip-Off integration alleles result in >99% reduction of gene expression in homozygotes and recapitulate known indel loss of function phenotypes. Passive, gene on orientation rbbp4-UFlip-On and rb1-UFlip-On integration alleles do not cause phenotypes in trans-heterozygous combination with an indel mutation. Cre recombinase injection leads to recombination at alternating pairs of loxP and lox2272 sites, inverting and locking the cassette into the active, gene off orientation, and the expected mutant phenotypes. In combination with our endogenous neural progenitor Cre drivers we demonstrate rbbp4-UFlip-On and rb1-UFlip-On gene inactivation phenotypes can be restricted to specific neural cell populations. Replacement of the UFlip mRFP primary reporter gene trap with a 2A-RFP in rbbp4-UFlip-Off, or 2A-KalTA4 in rb1-UFlip-Off, shows strong RFP expression in wild type or UAS:RFP injected embryos, respectively. Together these results validate a simplified approach for efficient isolation of highly mutagenic Cre/lox responsive conditional gene alleles to advance zebrafish Cre recombinase genetics.

genetics↗