A novel real-time PCR system for simultaneous detection of human viruses in clinical samples from patients with uncertain diagnoses

Abstract

A novel simultaneous detection system for human viruses was developed using a real-time polymerase chain reaction (PCR) system to identify causes of infection in clinical samples from patients with uncertain diagnoses. This system, designated as the "multivirus real-time PCR," has the potential to detect 163 human viruses (47 DNA viruses and 116 RNA viruses) in a 96-well plate simultaneously. The specificity and sensitivity of each probe-primer set were confirmed with cells or tissues infected with specific viruses. The multivirus real-time PCR system showed profiles of virus infection in 20 autopsies of acquired immunodeficiency syndrome patients, and detected frequently TT virus, cytomegalovirus, human herpesvirus 6, and Epstein-Barr virus in various organs; however, RNA viruses were detected rarely except for human immunodeficiency virus-1. Pathology samples from 40 patients with uncertain diagnoses were examined, including cases of encephalitis, hepatitis, and myocarditis. Herpes simplex virus 1, human herpesvirus 6, and parechovirus 3 were identified as causes of diseases in four cases of encephalitis, while no viruses were identified in other cases as causing disease. This multivirus real-time PCR system can be useful for detecting virus in specimens from patients with uncertain diagnoses.

Figure 1

Establishment and validation of multivirus real‐time PCR. A: Procedure of the multivirus real‐time PCR system. DNA sample was mixed with Quantitect 2× master mix, and RNA sample was mixed with Quantitect 2× master mix and reverse transcriptase (RT) mix. These mixtures were poured into each well in a 96‐well plate at 10 µl per well. Ten microliters of 2× probe and primer mix were then added to each well in a premixed 96‐well plate. Finally, the virus genes were amplified and detected in a real‐time PCR machine for 2 hr. B: Validation of the multivirus real‐time PCR. A gene expression image by TreeView software based on the results of the multivirus real‐time PCR for control samples is shown. A horizontal line shows each probe–primer set and a vertical line is one sample of positive control. Gray vertical lines indicate no sample. A scale bar indicates copy number of color. A green box indicates specific reactions of target positive controls in specific probe–primer sets. Arrows of (a–c) also show specific signals. The arrow (a) shows positive signal for TTV in a brain sample with both JCV and TTV infection. The arrows (b1–3) show that a probe–primer set for pan‐enterovirus reacted with poliovirus (b1), Coxsackievirus B3 (b2), and Echovirus 6 (b3) positive samples. The arrow (c) shows that a probe–primer set for influenza virus A reacted with H5N1 influenza virus. Details of positive controls were listed in Supplementary Table II.

Figure 2

Viruses detected in autopsied organs of AIDS patients. A: Positive number of each virus in the 20 cases of AIDS autopsy. B–D: Positive rates of CMV, HIV‐1, and HHV‐6 in organs. GI, gastrointestinal.

Figure 3

Mean values of virus copy numbers in organs. Mean of copy numbers per cells are shown in DNA samples (A–F). Mean ratios of virus copy number per hGAPDH‐RNA copy number are shown in RNA samples (G–I). Error bars show standard errors. One brain sample contained HIV‐associated encephalopathy (C,H), and one of the heart and adrenal sample contained EBV‐associated lymphoma (F).