scholarly journals Development of a real-time RT-PCR assay for the detection of pan-human parechoviruses

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Huanhuan Lu ◽  
Jinbo Xiao ◽  
Keyi Zhang ◽  
Zhenzhi Han ◽  
Yang Song ◽  
...  
Keyword(s):  
Real Time ◽  
Phylogenetic Analyses ◽  
Sequence Information ◽  
Healthy Children ◽  
Rt Pcr ◽  
Probe Sequence ◽  
Pcr Assay ◽  
Faecal Samples ◽  
Good Repeatability ◽  
Beijing China

Abstract Background 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. Methods 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). Results 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. Conclusion 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.

scholarly journals Development of a real-time RT-PCR assay for the detection of pan-human parechoviruses

2021 ◽  
Author(s):  
Huanhuan Lu ◽  
Jinbo Xiao ◽  
Keyi Zhang ◽  
Zhenzhi Han ◽  
Yang Song ◽  
...  
Keyword(s):  
Real Time ◽  
Phylogenetic Analyses ◽  
Sequence Information ◽  
Healthy Children ◽  
Rt Pcr ◽  
Probe Sequence ◽  
Pcr Assay ◽  
The Real ◽  
Good Repeatability ◽  
Beijing China

Abstract Background 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, only a few laboratories worldwide routinely conduct tests for the identification of this group of viruses; presently, to the best of our knowledge, no laboratory in China conducts routine test for PeV-A identification. Therefore, in this study, we aimed to develop and validate a real-time RT-PCR assay for the identification of PeV-As. Methods To designed 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). Results 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, the real-time RT-PCR assay showed good repeatability, reproducibility, and sensitivity. Conclusion Our findings suggested that the real-time RT-PCR assay developed in this study can be applied for routine PeV-A identification. 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.

scholarly journals Individual-based dengue virus surveillance in Aedes aegypti mosquitoes collected concurrently with suspected patients in Tarlac City, Philippines

2020 ◽  
Author(s):  
Jean Claude Balingit ◽  
Thaddeus M. Carvajal ◽  
Mariko Saito-Obata ◽  
Maribet Gamboa ◽  
Amalea Dulcene Nicolasora ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Dengue Virus ◽  
Vector Control ◽  
Real Time ◽  
Phylogenetic Analyses ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Virus Surveillance ◽  
One Step ◽  
Mosquito Infection

Abstract Background: Vector control measures are critical in the prevention and reduction of dengue virus (DENV) transmission. In this context, effective vector control is reliant not only on knowledge of mosquito abundance, but also on the timely and accurate detection of mosquito infection. Mosquito-based virus surveillance programs commonly rely on pool-based mosquito testing, but whether individual-based mosquito testing could represent a feasible alternative is not largely studied. Applying an individual-based mosquito testing approach, we conducted a one-month DENV surveillance of adult Aedes aegypti mosquitoes around households of suspected dengue patients during the 2015 dengue peak season in Tarlac City, Philippines to more accurately assess the mosquito infection rate, and to identify the DENV serotypes and genotypes concurrently co-circulating in mosquitoes and patients. Methods: We performed a one-step multiplex real-time RT-PCR assay for the simultaneous detection and serotyping of DENV in patients and in individual female Ae. aegypti mosquito. Additionally, we performed sequencing and phylogenetic analyses to further characterize the detected DENVs in mosquitoes and patients at the genotype level. Results: We collected a total of 583 adult Ae. aegypti mosquitoes, of which we tested 359 female mosquitoes individually for the presence of the DENV. Ten mosquitoes (2.8%) from amongst 359 female mosquitoes were confirmed to be positive for the presence of the DENV. We detected DENV-1, DENV-2, and DENV-4 in the field-collected mosquitoes, which were consistent with the serotypes concurrently infecting patients. Sequencing and phylogenetic analyses of the detected DENVs based on the partial envelope (E) gene revealed three genotypes concurrently present in the sampled mosquitoes and patients during the study period, namely: DENV-1 genotype IV, DENV-2 Cosmopolitan genotype and DENV-4 genotype II. Conclusions: In this study, we demonstrate the utility of a one-step multiplex real-time RT-PCR assay in individual-based DENV surveillance of mosquitoes. Our findings reinforce the importance of detecting and monitoring virus activity in local mosquito populations, which is critical for dengue prevention and control activities.

scholarly journals Surveillance of dengue virus in individual Aedes aegypti mosquitoes collected concurrently with suspected human cases in Tarlac City, Philippines

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Jean Claude Balingit ◽  
Thaddeus M. Carvajal ◽  
Mariko Saito-Obata ◽  
Maribet Gamboa ◽  
Amalea Dulcene Nicolasora ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Dengue Virus ◽  
Vector Control ◽  
Real Time ◽  
Phylogenetic Analyses ◽  
Simultaneous Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Denv Serotypes ◽  
One Step

Abstract Background Vector control measures are critical for the prevention and reduction of dengue virus (DENV) transmission. Effective vector control is reliant not only on knowledge of mosquito abundance, but also on the timely and accurate detection of mosquito-borne infection. Mosquito-based virus surveillance programs typically rely on pool-based mosquito testing, although whether individual-based mosquito testing is a feasible alternative to this has not been widely studied. Applying an individual-based mosquito testing approach, we conducted a 1-month surveillance study of DENV in adult Aedes aegypti mosquitoes in homes of suspected dengue patients during the 2015 peak dengue season in Tarlac City, Philippines to more accurately assess the mosquito infection rate and identify the DENV serotypes and genotypes concurrently co-circulating in mosquitoes and patients there. Methods We performed a one-step multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for the simultaneous detection and serotyping of DENV in patients and individual female Ae. aegypti mosquitoes. Additionally, we performed sequencing and phylogenetic analyses to further characterize the detected DENV serotypes in mosquitoes and patients at the genotype level. Results We collected a total of 583 adult Ae. aegypti mosquitoes, of which we individually tested 359 female mosquitoes for the presence of DENV. Ten (2.8%) of the 359 female mosquitoes were positive for the presence of DENV. We detected DENV-1, DENV-2, and DENV-4 in the field-collected mosquitoes, which was consistent with the serotypes concurrently found in infected patients. Sequencing and phylogenetic analyses of the detected DENV serotypes based on the partial sequence of the evelope (E) gene revealed three genotypes concurrently present in the sampled mosquitoes and patients during the study period, namely DENV-1 genotype IV, DENV-2 Cosmopolitan genotype, and DENV-4 genotype II. Conclusions We demonstrated the utility of a one-step multiplex real-time RT-PCR assay for the individual-based DENV surveillance of mosquitoes. Our findings reinforce the importance of detecting and monitoring virus activity in local mosquito populations, which are critical for dengue prevention and control.

scholarly journals Individual-based dengue virus surveillance in Aedes aegypti mosquitoes concurrently collected with suspected patients in Tarlac City, Philippines

2020 ◽  
Author(s):  
Jean Claude Balingit ◽  
Thaddeus M. Carvajal ◽  
Mariko Saito-Obata ◽  
Maribet Gamboa ◽  
Amalea Dulcene Nicolasora ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Vector Control ◽  
Real Time ◽  
Phylogenetic Analyses ◽  
Control Measures ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Virus Surveillance ◽  
One Step ◽  
Mosquito Infection

Abstract Background: Vector control measures are critical in the prevention and reduction of dengue virus (DENV) transmission. In this context, an effective vector control is reliant not only on the knowledge of mosquito abundance, but also on the timely and accurate detection of mosquito infection. Mosquito-based virus surveillance programs typically rely on a pool-based mosquito testing, although whether individual-based mosquito testing could represent a feasible alternative is not largely studied. Applying an individual-based mosquito testing approach, we conducted a one-month DENV surveillance of adult Aedes (Ae.) aegypti mosquitoes around households of suspected dengue patients during the 2015 dengue peak season in Tarlac City, Philippines to more accurately assess the mosquito infection rate and identify the DENV serotypes and genotypes concurrently co-circulating in mosquitoes and patients.Methods: We performed a one-step multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for the simultaneous DENV detection and serotyping in patients and individual female Ae. aegypti mosquito. Additionally, we performed sequencing and phylogenetic analyses to further characterize the detected DENVs in mosquitoes and patients at the genotype level.Results: We collected a total of 583 adult Ae. aegypti mosquitoes; of which, we individually tested 359 female mosquitoes for the presence of DENV. Ten (2.8%) among the 359 female mosquitoes were positive for the presence of DENV. We detected DENV-1, DENV-2, and DENV-4 in the field-collected mosquitoes, which were consistent with the serotypes concurrently infecting patients. Sequencing and phylogenetic analyses of the detected DENVs based on the partial envelope (E) gene revealed three genotypes concurrently present in the sampled mosquitoes and patients during the study period, namely: DENV-1 genotype IV, DENV-2 Cosmopolitan genotype, and DENV-4 genotype II.Conclusions: In this study, we demonstrate the utility of the one-step multiplex real-time RT-PCR assay in the individual-based DENV surveillance of mosquitoes. Our findings reinforce the significance of detecting and monitoring virus activity in local mosquito populations, which is critical for dengue prevention and control activities.

scholarly journals Multiple Types of Novel Enteric Bopiviruses (Picornaviridae) with the Possibility of Interspecies Transmission Identified from Cloven-Hoofed Domestic Livestock (Ovine, Caprine and Bovine) in Hungary

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 66
Author(s):  
Zoltán László ◽  
Péter Pankovics ◽  
Gábor Reuter ◽  
Attila Cságola ◽  
Ádám Bálint ◽  
...  
Keyword(s):  
Novel Species ◽  
Phylogenetic Analyses ◽  
Interspecies Transmission ◽  
Background Information ◽  
Type Ii ◽  
Rt Pcr ◽  
Faecal Samples ◽  
The Family ◽  
Domestic Livestock

Most picornaviruses of the family Picornaviridae are relatively well known, but there are certain “neglected” genera like Bopivirus, containing a single uncharacterised sequence (bopivirus A1, KM589358) with very limited background information. In this study, three novel picornaviruses provisionally called ovipi-, gopi- and bopivirus/Hun (MW298057-MW298059) from enteric samples of asymptomatic ovine, caprine and bovine respectively, were determined using RT-PCR and dye-terminator sequencing techniques. These monophyletic viruses share the same type II-like IRES, NPGP-type 2A, similar genome layout (4-3-4) and cre-localisations. Culture attempts of the study viruses, using six different cell lines, yielded no evidence of viral growth in vitro. Genomic and phylogenetic analyses show that bopivirus/Hun of bovine belongs to the species Bopivirus A, while the closely related ovine-origin ovipi- and caprine-origin gopivirus could belong to a novel species “Bopivirus B” in the genus Bopivirus. Epidemiological investigation of N = 269 faecal samples of livestock (ovine, caprine, bovine, swine and rabbit) from different farms in Hungary showed that bopiviruses were most prevalent among <12-month-old ovine, caprine and bovine, but undetectable in swine and rabbit. VP1 capsid-based phylogenetic analyses revealed the presence of multiple lineages/genotypes, including closely related ovine/caprine strains, suggesting the possibility of ovine–caprine interspecies transmission of certain bopiviruses.

scholarly journals Development and application of a real-time RT-PCR assay to rapidly detect H2 subtype avian influenza A viruses

2021 ◽  
pp. 104063872199481
Author(s):  
Yixin Xiao ◽  
Fan Yang ◽  
Fumin Liu ◽  
Hangping Yao ◽  
Nanping Wu ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Real Time ◽  
Influenza A ◽  
Minor Groove Binder ◽  
Rt Pcr ◽  
Influenza A Viruses ◽  
Pcr Assay ◽  
Infectious Dose ◽  
The Matrix ◽  
Taqman Minor Groove Binder

The H2 subtypes of avian influenza A viruses (avian IAVs) have been circulating in poultry, and they have the potential to infect humans. Therefore, establishing a method to quickly detect this subtype is pivotal. We developed a TaqMan minor groove binder real-time RT-PCR assay that involved probes and primers based on conserved sequences of the matrix and hemagglutinin genes. The detection limit of this assay was as low as one 50% egg infectious dose (EID50)/mL per reaction. This assay is specific, sensitive, and rapid for detecting avian IAV H2 subtypes.

A one-step multiplex real-time RT-PCR assay for rapid and simultaneous detection of human norovirus genogroup I, II and IV

2013 ◽  
Vol 189 (2) ◽  
pp. 277-282 ◽  
Author(s):  
Yong Yan ◽  
Heng-hui Wang ◽  
Lei Gao ◽  
Ji-mei Ji ◽  
Zhi-jie Ge ◽  
...  
Keyword(s):  
Real Time ◽  
Simultaneous Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Human Norovirus ◽  
One Step

Multicenter evaluation of the ENTEROVIRUS R-gene™ real-time RT-PCR assay for the detection of enteroviruses in clinical specimens

2010 ◽  
Vol 47 (1) ◽  
pp. 54-59 ◽  
Author(s):  
Sylvie Pillet ◽  
Geneviève Billaud ◽  
Shabir Omar ◽  
Bruno Lina ◽  
Bruno Pozzetto ◽  
...  
Keyword(s):  
Real Time ◽  
R Gene ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Clinical Specimens

scholarly journals Monitoring Current-Season Spread of Potato virus Y in Potato Fields Using ELISA and Real-Time RT-PCR

Plant Disease ◽  
2013 ◽  
Vol 97 (5) ◽  
pp. 641-644 ◽  
Author(s):  
Manphool S. Fageria ◽  
Mathuresh Singh ◽  
Upeksha Nanayakkara ◽  
Yvan Pelletier ◽  
Xianzhou Nie ◽  
...  
Keyword(s):  
Real Time ◽  
Potato Virus ◽  
Potato Virus Y ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Current Season ◽  
Seed Market ◽  
Polymerase Chain ◽  
Time Of Harvest

The current-season spread of Potato virus Y (PVY) was investigated in New Brunswick, Canada, in 11 potato fields planted with six different cultivars in 2009 and 2010. In all, 100 plants selected from each field were monitored for current-season PVY infections using enzyme-linked immunosorbent assay (ELISA) and real-time reverse-transcription polymerase chain reaction (RT-PCR) assay. Average PVY incidence in fields increased from 0.6% in 2009 and 2% in 2010 in the leaves to 20.3% in 2009 and 21.9% in 2010 in the tubers at the time of harvest. In individual fields, PVY incidence in tubers reached as high as 37% in 2009 and 39% in 2010 at the time of harvest. Real-time RT-PCR assay detected more samples with PVY from leaves than did ELISA. A higher number of positive samples was also detected with real-time RT-PCR from growing tubers compared with the leaves collected from the same plant at the same sampling time. PVY incidence determined from the growing tubers showed a significant positive correlation with the PVY incidence of tubers after harvest. Preharvest testing provides another option to growers to either top-kill the crop immediately to secure the seed market when the PVY incidence is low or leave the tubers to develop further for table or processing purposes when incidence of PVY is high.

Sign in / Sign up

Export Citation Format

Share Document