Rapid Detection of Novel Influenza A Virus and Seasonal Influenza A (H1N1, H3N2) Viruses by Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP)

Abstract

Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay we developed detects novel influenza A (H1N1) of swine origin and seasonal influenza A (H1N1 and H3N2) viruses. Individual primer sets targeting the HA gene for novel H1N1, H1N1, and H3N2 were newly designed to specifically detect these subtypes. No cross-reactions occurred among novel H1N1, H1N1, and H3N2, and 7 respiratory viruses-influenza B virus, influenza C virus, adenovirus, respiratory syncytial virus, metapneumovirus, parainfluenza virus, and rhinovirus-had no reaction to 3 RT-LAMP assays. RT-LAMP is assayed at 63 degrees C for 40 min. In our RT-LAMP assay, Eriochrome Black T was added to the reaction mixture as an amplification indicator to detect virus genomes without using real-time turbidimetry. Positive reactions were indicated in blue and negative reactions remained purple. Of 139 samples from suspected novel H1N1 subjects tested by both RT-LAMP and real-time RT-PCR assay, 110 were positive in both assays. Two samples with low copy numbers were positive only in real-time RT-PCR assay. Of 27 novel negative H1N1 samples, 4 were positive for H3N2 on viral isolation and conventional RT-PCR assay. RT-LAMP assay for detecting H3N2 obtained the same findings. Our RT-LAMP assay is thus potentially useful in rapidly detecting influenza A virus such as novel H1N1, H1N1, and H3N2.

A LAMP Protocol for the Detection of ‘Candidatus Phytoplasma pyri’, the Causal Agent of Pear Decline

“…At a pH between 7 and 11, the dye is in its blue form in the absence of metal ions and turns to a bright purple when it forms a complex with magnesium (Skoog et al 2014). Until now, the use of ErioT in LAMP reactions was only rarely reported; however, Shigemoto et al (2010), Oh et al (2016), andRodriguez-Manzano et al (2016) demonstrated the suitability of ErioT for colorimetric detection in LAMP assays.…”

Rapid and simple colorimetric detection of multiple influenza viruses infecting humans using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform

“…Our multiplex RT-LAMP detection system was designed to be a more feasible RIDT with accuracy as high as RT-PCR methods for detecting most recent human influenza viruses and avian influenza viruses infecting humans. Although a variety of reliable and affordable RT-LAMP methods have been developed to detect human or avian influenza viruses, each can only detect an individual subtype [23-29, 34, 35] or limited subtypes of human influenza viruses and/or avian influenza viruses infecting humans [18,30,36,37]. In a pandemic, it is critical to rapidly differentiate patients infected by seasonal flu from those infected by an emerging strain.…”

“…The specificity and sensitivity of RT-LAMP are potentially comparable to those of existing PCR-based diagnostics with much shorter reaction time. Recently, influenza diagnostics tools leveraging RT-LAMP technology have been reported [23][24][25][26][27][28][29][30]. However, most of these tools can only diagnose a single or a small number of human influenza viruses.…”

Aptasensors for viral diagnostics

“…Determining the cause of infection solely based on clinical parameters is quite impossible, while the treatment of viral and bacterial infections differs significantly. Assay requirements for viral diagnostics, such as sensitivity and the possibility High rate of false positives [11,12] NA, Not available; EID50, 50% egg infective dose -1 EID50 is the amount of virus that will infect 50% of inoculated eggs; VP, Virus particles; HAU, Hemagglutination units -1 HAU is the amount of virus needed to agglutinate an equal volume of standardized red blood cells. for multiplexing and quantification all depend on the purpose of the application.…”