Multiplexed Reverse Transcription Loop-Mediated Isothermal Amplification Coupled with a Nucleic Acid-Based Lateral Flow Dipstick as a Rapid Diagnostic Method to Detect SARS-CoV-2.

Due to the high reproduction rate of COVID-19, it is important to identify and isolate infected patients at the early stages of infection. The limitations of current diagnostic methods are speed, cost, and accuracy. Furthermore, new viral variants have emerged with higher rates of infectivity and mortality, many with mutations at various primer binding sites, which may evade detection via conventional PCR kits. Therefore, a rapid method that is sensitive, specific, and cost-effective is needed for a point-of-care molecular test. Accordingly, we developed a rapid molecular SARS-CoV-2 detection kit with high specificity and sensitivity, RT-PCR, taking advantage of the loop-mediated isothermal amplification (LAMP) technique. Four sets of six primers were designed based on conserved regions of the SARS-CoV-2 genome: two outer, two inner and two loop primers. Using the optimized protocol, SARS-CoV-2 genes were detected as quickly as 10 min but were most sensitive at 30 min, detecting as little as 100 copies of template DNA. We then coupled the RT-LAMP with a lateral flow dipstick (LFD) for multiplex detection. The LFD could detect two genic amplifications on a single strip, making it suitable for multiplexed detection. The development of a multiplexed RT-LAMP-LFD reaction on crude VTM samples would be suitable for the point-of-care diagnosis of COVID-19 in diagnostic laboratories as well as in private homes.

Allele mining, amplicon sequencing and computational prediction of Solanum melongena L. FT/TFL1 gene homologs uncovers putative variants associated to seed dormancy and germination.

The FT/TFL1 gene homolog family plays a crucial role in the regulation of floral induction, seed dormancy and germination in angiosperms. Despite its importance, the FT/TFL1 gene homologs in eggplant (Solanum melongena L.) have not been characterized to date. In this study, we performed a genome-wide identification of FT/TFL1 genes in eggplant using in silico genome mining. The presence of these genes was validated in four economically important eggplant cultivars (Surya, EP-47 Annamalai, Pant Samrat and Arka Nidhi) through Pacbio RSII amplicon sequencing. Our results revealed the presence of 12 FT/TFL1 gene homologs in eggplant, with evidence of diversification among FT-like genes suggesting their possible adaptations towards various environmental stimuli. The amplicon sequencing also revealed the presence of two alleles for certain genes (SmCEN-1, SmCEN-2, SmMFT-1 and SmMFT-2) of which SmMFT-2 was associated with seed dormancy and germination. This association was further supported by the observation that seed dormancy is rarely reported in domesticated eggplant cultivars, but is commonly observed in wild species. A survey of the genetic regions in domesticated cultivars and a related wild species, S. incanum, showed that the alternative allele of S. incanum was present in some members of the Pant Samrat cultivar, but was absent in most other cultivars. This difference could contribute to the differences in seed traits between wild and domesticated eggplants.

High Prevalence of Human Papillomavirus Types 56 and 70 Identified in the Native Populations of Sabah, Malaysia

Background: Cervical cancer is currently the third most common female cancer in Malaysia , with the human papillomavirus (HPV) considered as one of the important contributory factors. This study was conducted to determine HPV prevalence, its genotype distribution, and other potential risk factors among women in Kota Kinabalu, Sabah in order to evaluate the likely efficacy of current HPV vaccines in the local population. Methods: A total of 240 cervical samples were collected and subjected to DNA extraction, PCR amplification using the MY09/MY11 primer pair, and restriction fragment length polymorphism (RFLP) for HPV detection and genotyping. Sociodemographic, clinical, and behavioural data were also collected via questionnaires. Results: The prevalence of HPV infection was 9.6%. The most common HPVs among 13 genotypes were high-risk HPV-56 (16.7%) and probable high-risk HPV-70 (16.7%) followed by HPV-16, -58, -53, -61, -33, -59, and -66 (in decreasing order of prevalence) including the rare genotypes: HPV-62, -81, -82 and -84. Statistical analyses using logistic regression models showed that HPV infection was significantly associated with employment (OR 4.94; CI 1.58-15.40) and education at secondary/high school level (OR 0.13; CI 0.03-0.62). Conclusion: Distribution of HPV genotypes in Sabah indicated a high prevalence of HPV-56 and -70 which are among the rare HPV types in West Malaysia and merit consideration in future strategies for HPV vaccination specifically for local Sabahan women.

De novo assembly, characterization and functional annotation of pineapple fruit transcriptome through massively parallel sequencing.

BACKGROUND: Pineapple (Ananas comosus var. comosus), is an important tropical non-climacteric fruit with high commercial potential. Understanding the mechanism and processes underlying fruit ripening would enable scientists to enhance the improvement of quality traits such as, flavor, texture, appearance and fruit sweetness. Although, the pineapple is an important fruit, there is insufficient transcriptomic or genomic information that is available in public databases. Application of high throughput transcriptome sequencing to profile the pineapple fruit transcripts is therefore needed. METHODOLOGY/PRINCIPAL FINDINGS: To facilitate this, we have performed transcriptome sequencing of ripe yellow pineapple fruit flesh using Illumina technology. About 4.7 millions Illumina paired-end reads were generated and assembled using the Velvet de novo assembler. The assembly produced 28,728 unique transcripts with a mean length of approximately 200 bp. Sequence similarity search against non-redundant NCBI database identified a total of 16,932 unique transcripts (58.93%) with significant hits. Out of these, 15,507 unique transcripts were assigned to gene ontology terms. Functional annotation against Kyoto Encyclopedia of Genes and Genomes pathway database identified 13,598 unique transcripts (47.33%) which were mapped to 126 pathways. The assembly revealed many transcripts that were previously unknown. CONCLUSIONS: The unique transcripts derived from this work have rapidly increased of the number of the pineapple fruit mRNA transcripts as it is now available in public databases. This information can be further utilized in gene expression, genomics and other functional genomics studies in pineapple.