A highly sensitive one-tube nested quantitative real-time PCR assay for specific detection of Bordetella pertussis using the LNA technique.

Objectives: Bordetella pertussis is a highly contagious respiratory agent and is the causative pathogen of pertussis, which primarily affects children. Current diagnostic techniques for this pathogen have a variety of limitations including a long culture time, low bacterial load, and lack of specificity.

Methods: This article reports the development of a one-tube nested quantitative real-time PCR assay using the locked nucleic acid (LNA) technique (LNA-OTN-q-PCR), targeting the BP485 gene and using a simple inexpensive extraction method.

Development of an Internally Controlled Reverse Transcription Recombinase-aided Amplification Assay for the Rapid and Visual Detection of West Nile Virus.

West Nile virus (WNV) causes West Nile fever and West Nile encephalitis. Because infection by WNV creates serious public health problems, its simple, rapid, and visual detection is very important in clinical practice, especially in resource-limited laboratories. We have developed a rapid, specific, and highly sensitive internally controlled reverse transcription recombinase-aided amplification (RTRAA) assay to detect WNV, using both real-time fluorescence and the lateral flow dipstick (LFD) at 39.

Applicability of duplex real time and lateral flow strip reverse-transcription recombinase aided amplification assays for the detection of Enterovirus 71 and Coxsackievirus A16.

Background: Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two main etiological agents of Hand, Foot and Mouth Disease (HFMD). Simple and rapid detection of EV71 and CA16 is critical in resource-limited settings.

Methods: Duplex real time reverse-transcription recombinase aided amplification (RT-RAA) assays incorporating competitive internal amplification controls (IAC) and visible RT-RAA assays combined with lateral flow strip (LFS) for detection of EV71 and CA16 were developed respectively.

A rapid and sensitive recombinase aided amplification assay incorporating competitive internal control to detect Bordetella pertussis using the DNA obtained by boiling.

Objectives: Pertussis is a highly transmissible acute respiratory infection caused by the bacterial pathogen Bordetella pertussis. The purpose of this study was to develop a rapid, simple and sensitive diagnostic test for detecting this pathogen.

Methods: Here we present a recombinase aided amplification (RAA) assay incorporating competitive internal amplification control (IAC) to detect Bordetella pertussis using the DNA obtained by boiling.

Development and evaluation of recombinase-aided amplification assays incorporating competitive internal controls for detection of human adenovirus serotypes 3 and 7.

Background: Human adenoviruses are a common group of viruses that cause acute infectious diseases. Human adenovirus (HAdV) 3 and HAdV 7 cause major outbreaks of severe pneumonia. A reliable and practical method for HAdV typing in clinical laboratories is lacking.

Development of a reverse transcription recombinase-aided amplification assay for the detection of coxsackievirus A10 and coxsackievirus A6 RNA.

Hand, foot and mouth disease (HFMD) is a serious public health problem, and coxsackievirus A6 (CVA6) and coxsackievirus A10 (CVA10) are two of the major causative pathogens, in addition to enterovirus 71 (EV71) and coxsackievirus A16 (CVA16). A simple and rapid reverse transcription recombinase-aided amplification assay (RT-RAA) was developed for the detection of CVA10 and CVA6 in this study. The analytical sensitivity for detection of CVA10 and CVA6 at 95% probability by probit regression analysis was 35 copies per reaction and 38 copies per reaction, respectively, with 100% specificity.

An Improved Barcoded Oligonucleotide Primers-based Next-generation Sequencing Approach for Direct Identification of Viral Pathogens in Clinical Specimens.

Objective: To provide a feasible and cost-effective next-generation sequencing (NGS) method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use of common NGS, such as high cost, complicated procedures, tremendous data analysis, and high background noise in clinical samples.

Methods: Viruses from cell culture materials or clinical specimens were identified following an improved NGS procedure: reduction of background noise by sample preprocessing, viral enrichment by barcoded oligonucleotide (random hexamer or non-ribosomal hexanucleotide) primer-based amplification, fragmentation-free library construction and sequencing of one-tube mixtures, as well as rapid data analysis using an in-house pipeline.

Results: NGS data demonstrated that both barcoded primer sets were useful to simultaneously capture multiple viral pathogens in cell culture materials or clinical specimens and verified that hexanucleotide primers captured as many viral sequences as hexamers did.

Reverse transcription genome exponential amplification reaction assay for rapid and universal detection of human rhinoviruses.

Human rhinoviruses (HRVs) have long been recognized as the cause of more than one-half of acute viral upper respiratory illnesses, and they are associated with more-serious diseases in children, such as asthma, acute otitis media and pneumonia. A rapid and universal test for of HRV infection is in high demand. In this study, a reverse transcription genome exponential amplification reaction (RT-GEAR) assay targeting the HRV 5' untranslated region (UTR) was developed for pan-HRV detection.