Anchored Pan Dengue RT‐PCR and Fast Sanger sequencing for detection of Dengue RNA in human serum

Abstract

A large number of human infections are caused by different dengue virus strains, mainly in the tropical and subtropical parts of the world, but also outside the endemic regions. RT‐PCR methods are used widely for detection of dengue virus RNA in acute‐phase serum samples; however, new sequence variation can inhibit these methods. An assay was developed integrating an anchored Pan Dengue RT‐PCR with a new Fast Sanger sequencing protocol. For broad detection and identification of dengue virus RNA, including new strains of all serotypes, the conserved 3′ genome end was targeted for highly specific cDNA synthesis. A combination of degenerated primers was used for second strand synthesis, followed by tag primed amplification. The mixture of generated amplicons was identified directly by the Fast Sanger sequencing from the anchored 3′ genome end. Evaluating the assay on human serum RNA spiked with viral RNA representing the four dengue serotypes demonstrated a detection limit of 44–124 copies viral RNA per reaction for a two‐step format of the anchored Pan Dengue RT‐PCR and 100–500 copies for a one‐step protocol, respectively. The different serotypes were clearly identified from the generated sequences. Further, the 5‐hr procedure was evaluated and compared to standard real‐time RT‐PCR protocols on acute‐phase serum samples from patients with confirmed dengue infections. This assay demonstrates a strategy for virus detection, which combines nucleic acid amplification adapted for dengue virus RNA with direct and rapid sequencing. It provides a tolerance for new sequence variation and the strategy should be applicable for other RNA viruses. J. Med. Virol. 82:1701–1710, 2010. © 2010 Wiley‐Liss, Inc.

DNA Origami-Enabled Gene Localization of Repetitive Sequences

“…Organisms exhibit a wide range of traits determined by thousands of genes that are clustered on chromosomes carrying genetic information. Acquired and congenital gene variations (e.g., single nucleotide polymorphisms, complex gene structural variation) will threaten genomic stability, which eventually leads to genetic diseases. − Among them, the abnormal expansion of DNA repetitive sequences can cause more than 50 intractable human diseases (e.g., frontotemporal dementia, Huntington’s disease). , Typical methods for the detection of DNA repetitive sequences are sequencing and optical imaging-based technologies. − In 1990, Human Genome Project was implemented, aiming to determine the DNA sequence involved in the human chromosome and map the human genome for an in-depth understanding of gene localization and function. − This project has revolutionized sequencing technologies, which gives rise to the emergence of diverse methods for gene localization, such as Sanger sequencing technology, − Illumina sequencing technology, − and nanopore sequencing technology. − Each of these sequencing technologies has its application scenarios because of its own characteristics: Sanger sequencing for short read lengths, high accuracy, reliability, and controllability; Illumina sequencing for short read lengths, high throughput, high sensitivity, and suitability for large-scale sequencing; and nanopore sequencing for long read lengths, real-time sequencing, and portability. − …”

Ultra-sensitive chemiluminescence imaging DNA hybridization method in the detection of mosquito-borne viruses and parasites

“…Efficient prevention, control, and treatment of mosquito-borne diseases rely on the rapid detection of the mosquito-borne pathogens and accurate diagnosis. Currently, methods for detecting MBVs and MBPs are mainly based on genetic assays, which include real-time PCR [ 21 – 25 ], isothermal amplification [ 26 ], oligonucleotide microarray [ 27 ] and sequencing [ 28 ]. However, these methods were designed for detecting only certain species of MBVs or MBPs.…”