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. 2022 Jan 14;12(1):196.
doi: 10.3390/diagnostics12010196.

Analysis Comparison for Rapid Identification of Pathogenic Virus from Infected Tissue Samples

Junji Hosokawa-Muto  1 , Yukiko Sassa-O'Brien  2 , Yoshihito Fujinami  1 , Hiroaki Nakahara  1
Affiliations

Analysis Comparison for Rapid Identification of Pathogenic Virus from Infected Tissue Samples

Junji Hosokawa-Muto et al. Diagnostics (Basel). .

Abstract

When examining infectious samples, rapid identification of the pathogenic agent is required for diagnosis and treatment or for investigating the cause of death. In our previous study, we applied exhaustive amplification using non-specific primers (the rapid determination system of viral genome sequences, the RDV method) to identify the causative virus via swab samples from a cat with a suspected viral infection. The purpose of the current study is to investigate suitable methods for the rapid identification of causative pathogens from infected tissue samples. First, the influenza virus was inoculated into mice to prepare infected tissue samples. RNA extracted from the mouse lung homogenates was transcribed into cDNA and then analyzed using the RDV method and next-generation sequencing, using MiSeq and MinION sequencers. The RDV method was unable to detect the influenza virus in the infected tissue samples. However, influenza virus reads were detected using next-generation sequencing. Comparing MiSeq and MinION, the time required for library and sequence preparation was shorter for MinION sequencing than for MiSeq sequencing. We conclude that when a causative virus needs to be rapidly identified from an infectious sample, MinION sequencing is currently the method of choice.

Keywords: detection; exhaustive gene amplification; forensic; identification; infectious disease; next-generation sequencing; quantitative PCR; read; tissue sample; virus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Positions of amplicons on the influenza A virus PB1 gene. The 742 bp amplicon was used as a template for the standard curve.
Figure 2
Figure 2
Quantitation of the influenza A virus PB1 gene in the lung homogenate samples. Quantification was performed twice per sample, and the average of the two samples was calculated. Error bars indicate the standard deviations.
Figure 3
Figure 3
Partial electrophoretic results after amplification of the second library during the RDV method. In this amplification, H1–3 were used as forward primers, and H9–5 to H9–12 were used as reverse primers. Lane M, FlashGel™ DNA Marker, 50 bp–1.5 kb.
See this image and copyright information in PMC

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