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 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 Description and Validation of a Novel Real-Time RT-PCR Enterovirus Assay

2008 ◽  
Vol 54 (2) ◽  
pp. 406-413 ◽  
Author(s):  
Weston C Hymas ◽  
Wade K Aldous ◽  
Edward W Taggart ◽  
Jeffery B Stevenson ◽  
David R Hillyard
Keyword(s):  
Sequence Analysis ◽  
Single Nucleotide Polymorphisms ◽  
Real Time ◽  
Clinical Samples ◽  
Nucleotide Polymorphisms ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Single Nucleotide ◽  
The Real ◽  
Nontranslated Region

Abstract Background: Enteroviruses are a leading cause of aseptic meningitis in adult and pediatric populations. We describe the development of a real-time RT-PCR assay that amplifies a small target in the 5′ nontranslated region upstream of the classical Rotbart enterovirus amplicon. The assay includes an RNA internal control and incorporates modified nucleotide chemistry. Methods: We evaluated the performance characteristics of this design and performed blinded parallel testing on clinical samples, comparing the results with a commercially available RT-PCR assay (Pan-Enterovirus OligoDetect kit) that uses an enzyme immunoassay–like plate end detection. Results: We tested 778 samples and found 14 discrepant samples between the 2 assays. Of these, the real-time assay detected 6 samples that were negative by the OligoDetect kit, 5 of which were confirmed as positive by sequence analysis using an alternative primer set. Eight discrepant samples were positive by the OligoDetect kit and real-time negative, with 6 confirmed by sequencing. Overall, detection rates of 97% and 96% were obtained for the OligoDetect kit and real-time assays, respectively. Sequence analysis revealed the presence of a number of single nucleotide polymorphisms in the targeted region. The comparative sensitivities of the 2 assays were equivalent, with the limit of detection for the real-time assay determined to be approximately 430 copies per milliliter in cerebrospinal fluid. Conclusions: This novel real-time enterovirus assay is a sensitive and suitable assay for routine clinical testing. The presence of single nucleotide polymorphisms can affect real-time PCR assays.

scholarly journals Rapid SARS-CoV-2 antigen detection assay in comparison with real-time RT-PCR assay for laboratory diagnosis of COVID-19 in Thailand

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Chutikarn Chaimayo ◽  
Bualan Kaewnaphan ◽  
Nattaya Tanlieng ◽  
Niracha Athipanyasilp ◽  
Rujipas Sirijatuphat ◽  
...  
Keyword(s):  
Real Time ◽  
Sensitivity And Specificity ◽  
False Negative ◽  
Laboratory Diagnosis ◽  
Antigen Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Screening Assay ◽  
The Real ◽  
Pcr Test

Abstract Background The Coronavirus disease 2019 (COVID-19) pandemic continues to spread across the world. Hence, there is an urgent need for rapid, simple, and accurate tests to diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Performance characteristics of the rapid SARS-CoV-2 antigen detection test should be evaluated and compared with the gold standard real-time reverse transcription-polymerase chain reaction (RT-PCR) test for diagnosis of COVID-19 cases. Methods The rapid SARS-CoV-2 antigen detection test, Standard™ Q COVID-19 Ag kit (SD Biosensor®, Republic of Korea), was compared with the real-time RT-PCR test, Allplex™ 2019-nCoV Assay (Seegene®, Korea) for detection of SARS-CoV-2 in respiratory specimens. Four hundred fifty-four respiratory samples (mainly nasopharyngeal and throat swabs) were obtained from COVID-19 suspected cases and contact individuals, including pre-operative patients at Siriraj Hospital, Bangkok, Thailand during March–May 2020. Results Of 454 respiratory samples, 60 (13.2%) were positive, and 394 (86.8%) were negative for SARS-CoV-2 RNA by real-time RT-PCR assay. The duration from onset to laboratory test in COVID-19 suspected cases and contact individuals ranged from 0 to 14 days with a median of 3 days. The rapid SARS-CoV-2 antigen detection test’s sensitivity and specificity were 98.33% (95% CI, 91.06–99.96%) and 98.73% (95% CI, 97.06–99.59%), respectively. One false negative test result was from a sample with a high real-time RT-PCR cycle threshold (Ct), while five false positive test results were from specimens of pre-operative patients. Conclusions The rapid assay for SARS-CoV-2 antigen detection showed comparable sensitivity and specificity with the real-time RT-PCR assay. Thus, there is a potential use of this rapid and simple SARS-CoV-2 antigen detection test as a screening assay.

scholarly journals Primers and probe design and precision assessment of the real time RT-PCR assay in Coxsackievirus A10 and enterovirus detection

Data in Brief ◽  
2017 ◽  
Vol 12 ◽  
pp. 418-422
Author(s):  
Jingfang Chen ◽  
Rusheng Zhang ◽  
Xinhua Ou ◽  
Dong Yao ◽  
Zheng Huang ◽  
...  
Keyword(s):  
Real Time ◽  
Probe Design ◽  
Rt Pcr ◽  
Pcr Assay ◽  
The Real ◽  
Precision Assessment

scholarly journals Quantification of Substance P mRNA in Human Immune Cells by Real-Time Reverse Transcriptase PCR Assay

2002 ◽  
Vol 9 (1) ◽  
pp. 138-143 ◽  
Author(s):  
Jian-Ping Lai ◽  
Steven D. Douglas ◽  
Farida Shaheen ◽  
David E. Pleasure ◽  
Wen-Zhe Ho
Keyword(s):  
Substance P ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Immune Cells ◽  
Fetal Brain ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Pcr Assay ◽  
The Real ◽  
Human Immune

ABSTRACT We have applied a newly developed real-time reverse transcriptase (RT) PCR (RT-PCR) assay for quantification of substance P (SP) mRNA expression (the SP real-time RT-PCR assay) in human blood monocyte-derived macrophages, peripheral blood lymphocytes, and microglia isolated from fetal brain. The SP real-time RT-PCR assay had a sensitivity of 60 mRNA copies, with a dynamic range of detection between 60 and 600,000 copies of the SP gene transcript per reaction mixture. The coefficient of variation of the threshold cycle number between the SP real-time RT-PCR assays was less than 1.16%. This assay with an SP-specific primer pair efficiently recognizes all four isoforms of preprotachykinin A (the SP precursor) gene transcripts. In order to use this assay to measure the levels of SP mRNA in the human immune cells quantitatively, we designed a specific probe (molecular beacon) derived from exon 3 of the SP gene. We demonstrated that the real-time RT-PCR quantitatively detected SP mRNA in the human immune cells, among which the microglia isolated from fetal brain had the highest levels of SP mRNA. The SP real-time PCR assay yielded reproducible data, as the intra-assay variation was less than 1%. Thus, it is feasible to apply the real-time RT-PCR assay for quantification of SP mRNA levels in human immune cells, as well as in other nonneuronal cells. Since SP is a major modulator of neuroimmunoregulation, this assay has the potential for widespread application for basic and clinical investigations.

scholarly journals Combined Immunomagnetic Separation-Molecular Beacon-Reverse Transcription-PCR Assay for Detection of Hepatitis A Virus from Environmental Samples

2004 ◽  
Vol 70 (7) ◽  
pp. 4371-4374 ◽  
Author(s):  
Khaled H. Abd El Galil ◽  
M. A. El Sokkary ◽  
S. M. Kheira ◽  
Andre M. Salazar ◽  
Marylynn V. Yates ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Environmental Samples ◽  
Hepatitis A ◽  
Molecular Beacon ◽  
Hepatitis A Virus ◽  
Immunomagnetic Separation ◽  
Rt Pcr ◽  
Pcr Assay ◽  
The Real

ABSTRACT In this study, a molecular-beacon-based real-time reverse transcription (RT)-PCR assay was developed to detect the presence of hepatitis A virus (HAV) in environmental samples. A 125-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time RT-PCR assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, and only HAV was positively identified. When combined with immunomagnetic separation, the real-time RT-PCR assay successfully detected as few as 20 PFU in seeded groundwater samples. Because of its simplicity and specificity, this assay has broad applications for the rapid detection of HAV in contaminated foods or water.

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.

The utility of an investigational real-time RT-PCR assay system for the detection of micrometastases in sentinel lymph nodes of breast cancer confirmed by 0.2 mm histological interval analysis

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 10560-10560
Author(s):  
M. Kurozumi ◽  
Y. Kobayashi ◽  
H. Takei ◽  
K. Kitsugi ◽  
M. Ueno ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Lymph Node ◽  
Real Time ◽  
Interval Analysis ◽  
Histological Analysis ◽  
Frozen Section ◽  
Breast Cancer Patients ◽  
Rt Pcr ◽  
Pcr Assay ◽  
The Real

10560 Background: Comparison between real-time RT-PCR and histological analysis for the detection of lymph node metastasis in breast cancer patients is difficult because of the necessary use of different tissue for the two tests. In order to minimize this challenge this study used histology sections taken every 0.2 mm and all tissue between the sections was analyzed by real-time RT-PCR. Methods: 129 Lymph nodes (LNs) were obtained by Sentinel LN biopsy and/or axillary dissection from 80 breast cancer patients. All LNs were cut in half. One half of each LN was used for the routine intra-operative diagnosis. The other half was used for the 0.2mm histological interval analysis. All 10μm sections cut for histology at 0.2 mm intervals were analyzed using frozen section H&E under a light microscope. All remaining tissue between the sections used for histological analysis was assayed by real-time RT-PCR using the genetic markers mammaglobin and cytokeratin-19 (investigational GeneSearch Breast Lymph Node Assay, Veridex, LLC, Warren, NJ, USA). Cutoffs were pre- set for the real-time RT-PCR to detect only metastasis greater than 0.2 mm. Results: Compared to the histological diagnosis using 0.2 mm interval frozen sections, the real-time RT-PCR results were as follows; sensitivity of 100.0% (34/34), specificity of 93.7% (89/95) and overall accuracy of 95.3% (123/129). Conclusions: These data suggest by utilizing the 0.2 mm histological interval analysis, sampling discrepancies are minimized and a high level of sensitivity and overall accuracy is seen for the real-time RT-PCR assay. Assay specificity calculations may be less than 100% due to interpretation challenges in frozen section histology, especially in the cases of smaller metastases and/or lobular cancer, causing a small percentage of clinically relevant metastasis to be missed. [Table: see text]

scholarly journals Validation of Real-Time RT-PCR for Detection of SARS-CoV-2 in the Early Stages of the COVID-19 Outbreak Without Emergency Use Authorization Reagents in the Republic of Korea

2021 ◽  
Author(s):  
Yoon-Seok Chung ◽  
Nam-Joo Lee ◽  
Sang Hee Woo ◽  
Jeong-Min Kim ◽  
Heui Man Kim ◽  
...  
Keyword(s):  
Early Diagnosis ◽  
Real Time ◽  
Target Genes ◽  
Limit Of Detection ◽  
Republic Of Korea ◽  
Rt Pcr ◽  
Pcr Assay ◽  
The Real ◽  
Genomic Markers ◽  
The Republic

Abstract Background: A real-time reverse transcription polymerase chain reaction (RT-PCR) assay for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was developed to rapidly diagnose coronavirus disease 2019 (COVID-19). Early diagnosis of COVID-19 enables timely treatment and the implementation of public health measures. We validated the sensitivity, specificity, precision, linearity, accuracy, and robustness of a real-time RT-PCR-based assay for SARS-CoV-2 detection and compared the sensitivity and specificity of Emergency Use Authorization (EUA)-approved reagents for COVID-19 with those of the real-time RT-PCR method.Methods: The real-time RT-PCR-based assay was performed to specifically amplify genomic markers of SARS-CoV-2.Results: The real-time RT-PCR assay was highly specific for SARS-CoV-2, and did not amplify genomic fragments of 13 other viruses that cause respiratory diseases. The assay showed high linearity when conducted with a viral isolate from a patient with COVID-19 together with plasmids containing the target genes. The assay showed good repeatability and reproducibility, with a coefficient of variation of 3%, and detected SARS-CoV-2 with a limit of detection of 1 PFU/mL.Conclusions: The present real-time RT-PCR-based assay can diagnose COVID-19 with high accuracy and sensitivity. This approach is highly effective and can facilitate the early diagnosis of COVID-19 without the use of EUA reagents in the Republic of Korea.

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.

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