Performance of a real-time PCR-based approach and droplet digital PCR in detecting human parechovirus type 3 RNA.
Human parechovirus type 3 (HPeV3) is an emerging virus that causes sepsis and meningoencephalitis in neonates and young infants. Correct diagnosis of HPeV3 infection is critical in determining appropriate management and predicting patients' clinical course. Real-time reverse transcription PCR (RT-PCR) analysis of serum and/or cerebrospinal fluid (CSF) has been used to diagnose HPeV3 infection; however, the assay detection limits have not been fully evaluated.



We tested the hypothesis that droplet digital RT-PCR (RT-ddPCR)-a novel technique that precisely quantitates low-copy target genes by diluting and partitioning samples into compartments-increases the detection rate of HPeV3 RNA as compared with real-time RT-PCR.



Using samples with predetermined HPeV3 copy numbers, we evaluated one-step and two-step RT-ddPCR. Then, we tested two-step RT-ddPCR and real-time RT-PCR, using clinical samples with low copy numbers. Finally, we used two-step RT-ddPCR to evaluate clinical samples obtained from HPeV3-infected patients with positive serum but negative CSF, as determined by real-time RT-PCR.



Two-step RT-ddPCR was less variable and more specific than one-step RT-ddPCR. Two-step RT-ddPCR detected HPeV3 RNA in all six CSF samples; four samples (67%) were reproducibly positive and the other two samples (33%) were positive at least once in four replicates. Finally, no nonspecific droplet was positive by two-step RT-ddPCR.



Two-step RT-ddPCR may enhance the rate of HPeV3 RNA detection from samples with low viral loads, thereby improving diagnosis and management of HPeV3-infected patients.
Department of Pediatrics, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
Yuta
Y
Aizawa
Department of Molecular Neuroscience, Brain Research Institute, Niigata University, Niigata, Japan.
Akihide
A
Koyama
Department of Molecular Neuroscience, Brain Research Institute, Niigata University, Niigata, Japan.
Tomohiko
T
Ishihara
Department of Molecular Neuroscience, Brain Research Institute, Niigata University, Niigata, Japan.
Osamu
O
Onodera
Department of Pediatrics, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan. Electronic address: asaitoh@med.niigata-u.ac.jp.
Akihiko
A
Saitoh
D005260:Female; D005838:Genotype; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D057230:Limit of Detection; D008297:Male; D008590:Meningoencephalitis / Q000175:diagnosis / Q000821:virology; D030061:Parechovirus / Q000235:genetics* / Q000302:isolation & purification*; D010850:Picornaviridae Infections / Q000175:diagnosis* / Q000821:virology; D012367:RNA, Viral / Q000097:blood / Q000134:cerebrospinal fluid / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D018805:Sepsis / Q000175:diagnosis / Q000821:virology; D019562:Viral Load
D012367:RNA, Viral
Cerebrospinal fluid; Digital PCR; Human parechovirus type 3; Infants; Neonates; Real-time PCR