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Development of a real-time RT-PCR assay for the detection of pan-human parechoviruses. 2021

Huanhuan Lu, and Jinbo Xiao, and Keyi Zhang, and Zhenzhi Han, and Yang Song, and Dongyan Wang, and Tianjiao Ji, and Dongmei Yan, and Shuangli Zhu, and Wenbo Xu, and Yong Zhang
WHO WPRO Regional Polio Reference Laboratory and National Health Commission Key Laboratory of Biosafety, National Health Commission Key Laboratory of Medical Virology, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, People's Republic of China.

Parechoviruses (PeV-As), which constitute a new genus within the family Picornaviridae, have been associated with numerous localized outbreaks of serious diseases, such as coryza, pneumonia, maculopapular exanthem, and conjunctivitis. However, to the best of our knowledge, only a few laboratories worldwide conduct tests for the identification of this group of viruses. Therefore, in this study, we aimed to develop and validate a real-time RT-PCR assay for the identification of PeV-As. To design and validate a real-time PCR primer-probe targeting the 5'-UTR region of PeV-As, the 5'-UTR sequences of PeV-As available in GenBank were aligned using the MUSCLE algorithm in MEGA v7.0. Thereafter, the highly conserved 5'-UTR region was selected, and its primer-probe sequence was designed using Primer Premier v5.0. This primer-probe sequence was then evaluated for specificity, sensitivity, and repeatability, and for its validation, it was tested using fecal samples from 728 healthy children living in Beijing (China). The PeV-A real-time RT-PCR assay detected only the RNA-positive standards of PeV-A genotypes (1-8, 14, 17, and 18), whereas 72 serotypes of non-PeV-A EV viruses were undetected. In addition, the VP1 region of these 11 PeV-A genotypes that tested positive were amplified using the primers designed in this study. Typing results indicated that eight, one, and two strains of the 11 were PeV-A1, PeV-A4, and PeV-A6, respectively. We also determined and presented the genetic characterization and phylogenetic analyses results corresponding to these 11 VP1 region sequences. Furthermore, real-time RT-PCR assay showed good sensitivity with LOD of 102 copies/μL. Positive results in eight parallel experiments at each concentration gradient from 107 copies/μL to 102 copies/μL, indicating good repeatability. Our findings suggested that the real-time RT-PCR assay developed in this study can be applied for routine PeV-A identification. We detected PeV-A1, 4 and 6 genotypes in the 728 faecal samples using this method. Additionally, we believe that our results will serve as a foundation for further studies on PeV-As and facilitate the expansion of the gene sequence information available in GenBank.

UI MeSH Term Description Entries
D010802 Phylogeny The relationships of groups of organisms as reflected by their genetic makeup. Community Phylogenetics,Molecular Phylogenetics,Phylogenetic Analyses,Phylogenetic Analysis,Phylogenetic Clustering,Phylogenetic Comparative Analysis,Phylogenetic Comparative Methods,Phylogenetic Distance,Phylogenetic Generalized Least Squares,Phylogenetic Groups,Phylogenetic Incongruence,Phylogenetic Inference,Phylogenetic Networks,Phylogenetic Reconstruction,Phylogenetic Relatedness,Phylogenetic Relationships,Phylogenetic Signal,Phylogenetic Structure,Phylogenetic Tree,Phylogenetic Trees,Phylogenomics,Analyse, Phylogenetic,Analysis, Phylogenetic,Analysis, Phylogenetic Comparative,Clustering, Phylogenetic,Community Phylogenetic,Comparative Analysis, Phylogenetic,Comparative Method, Phylogenetic,Distance, Phylogenetic,Group, Phylogenetic,Incongruence, Phylogenetic,Inference, Phylogenetic,Method, Phylogenetic Comparative,Molecular Phylogenetic,Network, Phylogenetic,Phylogenetic Analyse,Phylogenetic Clusterings,Phylogenetic Comparative Analyses,Phylogenetic Comparative Method,Phylogenetic Distances,Phylogenetic Group,Phylogenetic Incongruences,Phylogenetic Inferences,Phylogenetic Network,Phylogenetic Reconstructions,Phylogenetic Relatednesses,Phylogenetic Relationship,Phylogenetic Signals,Phylogenetic Structures,Phylogenetic, Community,Phylogenetic, Molecular,Phylogenies,Phylogenomic,Reconstruction, Phylogenetic,Relatedness, Phylogenetic,Relationship, Phylogenetic,Signal, Phylogenetic,Structure, Phylogenetic,Tree, Phylogenetic
D010849 Picornaviridae A family of small RNA viruses comprising some important pathogens of humans and animals. Transmission usually occurs mechanically. There are nine genera: APHTHOVIRUS; CARDIOVIRUS; ENTEROVIRUS; ERBOVIRUS; HEPATOVIRUS; KOBUVIRUS; PARECHOVIRUS; RHINOVIRUS; and TESCHOVIRUS. Avihepatovirus,Pasivirus,Picornaviruses,Rosavirus,Sapelovirus,Senecavirus,Sicinivirus,Tremovirus,Avihepatoviruses,Pasiviruses,Rosaviruses,Sapeloviruses,Senecaviruses,Siciniviruses,Tremoviruses
D002648 Child A person 6 to 12 years of age. An individual 2 to 5 years old is CHILD, PRESCHOOL. Children
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D012367 RNA, Viral Ribonucleic acid that makes up the genetic material of viruses. Viral RNA
D012680 Sensitivity and Specificity Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed) Specificity,Sensitivity,Specificity and Sensitivity
D060888 Real-Time Polymerase Chain Reaction Methods used for detecting the amplified DNA products from the polymerase chain reaction as they accumulate instead of at the end of the reaction. Kinetic Polymerase Chain Reaction,Quantitative Real-Time PCR,Quantitative Real-Time Polymerase Chain Reaction,Real-Time PCR,PCR, Quantitative Real-Time,PCR, Real-Time,PCRs, Quantitative Real-Time,PCRs, Real-Time,Quantitative Real Time PCR,Quantitative Real Time Polymerase Chain Reaction,Quantitative Real-Time PCRs,Real Time PCR,Real Time Polymerase Chain Reaction,Real-Time PCR, Quantitative,Real-Time PCRs,Real-Time PCRs, Quantitative
D020133 Reverse Transcriptase Polymerase Chain Reaction A variation of the PCR technique in which cDNA is made from RNA via reverse transcription. The resultant cDNA is then amplified using standard PCR protocols. Polymerase Chain Reaction, Reverse Transcriptase,Reverse Transcriptase PCR,PCR, Reverse Transcriptase,Transcriptase PCR, Reverse
D030061 Parechovirus A genus in the family PICORNAVIRIDAE infecting humans and rodents. The type species is Human parechovirus. Human parechovirus,Ljungan virus,Echovirus 22,Echovirus 23,Human echovirus 22,Human echovirus 23,Human parechovirus 1,Human parechovirus 2,Human parechoviruses,Ljungan viruses,Parechoviruses,echovirus 23s, Human,parechovirus, Human

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