scholarly journals Quickly and simply detection for coronaviruses including SARS-CoV-2 on the mobile Real-Time PCR without treating RNA in advance

2020 ◽  
Author(s):  
Masaaki Muraoka ◽  
Yukiko Tanoi ◽  
Tetsutaro Tada ◽  
Mikio Mizukoshi ◽  
Osamu Kawaguchi
Keyword(s):  
Real Time ◽  
Correlation Coefficient ◽  
Real Time Pcr ◽  
Positive Control ◽  
List Type ◽  
Rt Pcr ◽  
Human Coronavirus ◽  
One Step ◽  
Short Time ◽  
Human Coronavirus 229E

ABSTRACTSARS-CoV-2 was reported to the WHO as an outbreak in Wuhan City, China on end of 2019, afterwards pandemic on the worldwide in 2020. The SARS-CoV-2 virus is less deadly, but far more transmissible. Therefore, it needs to detect and monitor quickly and simply on site to prevent SARS-CoV-2.If detecting coronaviruses including SARS-CoV-2, the real-time RT-PCR method is sensitive and specific for the unique target, however, it must take long time and labour that RNA is treated in advance, transcribed and amplified. Therefore, referenced previously report, in this study, we modified various methods to prove hypotheses the followed.Firstly, we hypothesized that real-time RT-PCR could be finished in very short time by the mobile real-time PCR device and one-step RT-PCR reagent. Secondly, we hypothesized that it was possible to perform RT-PCR utilizing the reagent as the above without RNA treatment in advance so called “direct”.Firstly, it was able to detect the positive control RNA of SARS-CoV-2 for less than 13.5 minutes by primer-probe referring to the CDC. Moreover, each detection value varied in accordance with each concentration (This correlation coefficient R2 > 0.95). Secondary, it was possible to detect human coronavirus 229E with direct RT-PCR. Furthermore, each detection value varied in accordance with each titer (TCID50 / mL) of human coronavirus 229E (This correlation coefficient R2 > 0.95).Considering the above, causing by utilizing the mobile real-time PCR device and the one-step real-time PCR reagent simultaneously following as: 1) It was possible to detect SARS-CoV-2 in very short time as compared to conventional method; 2) It was possible to detect human coronavirus quickly and simply with “direct”. For these reasons, we hypothesized that it is possible to detect SARS-CoV-2 quickly and simply by utilizing methods the above without treating RNA in advance. This hypothesis is our next try.STRENGTHS AND LIMITATIONS OF THIS STUDY*This study developed it possible to detect the positive control RNA of SARS-CoV-2 more quickly than previously, however couldn’t try to detect the genetic RNA.*This study proved clearly that the human coronavirus instead of SARS-CoV-2 could be detected simply without treating RNA in advance by the same method above.*This study couldn’t try to utilize the human specimens because of our institution limited.*This study could utilize the device and the reagents commercial and not especial.

scholarly journals Direct detection of Dengue viruses without extraction of RNA on the mobile real-time PCR device

2020 ◽  
Author(s):  
Masaaki Muraoka ◽  
Yukiko Tanoi ◽  
Tetsutaro Tada ◽  
Aya Tabata ◽  
Mikio Mizukoshi ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Direct Detection ◽  
Positive Control ◽  
Nucleic Acid Amplification ◽  
Severe Dengue ◽  
Mosquito Vectors ◽  
Rt Pcr ◽  
The Real ◽  
One Step

ABSTRACTDengue virus (DENV) is the cause of dengue / severe dengue and a virus of the Flaviviridae family, furthermore, dengue fever has rapidly spread in the world in recent decades. DENV is transmitted by female mosquitoes, mainly of the specie Aedes aegypti. The main method to control or prevent the transmission of DENV is to combat the mosquito vectors. Among these, one of important methods is to monitor the DENVs in the mosquito vectors.For the detection of DENV, nucleic acid amplification tests (NAAT) were recommended, of which criterion standard is real-time RT-PCR with highly sensitive and specific. However, it takes long time as to judge the result per a reaction, besides the necessity of the treatment of RNA in advance, example of extraction, concentration and purification.It was our object in this time to develop the method of real-time RT-PCR detecting DENVs in shorter time, moreover without especial treatment of RNA from the mosquito in advance. Besides, this work was performed with combing the mobile real-time PCR device with the one-step RT-PCR reagent.Firstly, we succeeded in shortening the time of real-time RT-PCR for the detection of DENV per one reaction, so that the judgement needed less than 20 minutes if genomic RNA treated in advance. Moreover, each value on the real-PCR device was quantitatively correlated with the positive control RNA from 1.0 × 10 ^ 3 copies to 1.0 × 10 ^ 0 copies per reaction (This correlation coefficient R2 > 0.95). Additionally, it made sure that this method could be applied to each DENV serotype.Secondly, we established the basis of procedure for the real-time RT-PCR without the treatment in advance so-called “direct”. As the result that the positive control RNA additive was utilized instead of the real DENV, spiked into the mosquito homogenized and sampled the supernatant without treatment, it was possible to detect on the real-time RT-PCR even if mosquitoes immediately after blood-feeding. For this reason, this method might be able to utilize in human sera, too.According to the results of this work, we could suggest the method is possible to detect DENV more quickly and more simply than heretofore. The Real-time “direct” RT-PCR, especially, could be performed with mobile real-time PCR PCR1100 device and one step RT-PCR reagent only. This method must help to detect some viruses other than DENV, too.

scholarly journals Comparison of a one-step real-time RT-PCR and a nested real-time RT-PCR for a genogroup II norovirus reveals differences in sensitivity depending upon assay design and visualization

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0248581
Author(s):  
Clyde S. Manuel ◽  
Cassandra Suther ◽  
Matthew D. Moore ◽  
Lee-Ann Jaykus
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Blot Hybridization ◽  
Molecular Techniques ◽  
Rt Pcr ◽  
Dot Blot ◽  
Dot Blot Hybridization ◽  
One Step

Human norovirus (NoV) is the leading cause of acute viral gastroenteritis and a major source of foodborne illness. Detection of NoV in food and environmental samples is typically performed using molecular techniques, including real-time reverse transcription polymerase chain reaction (RT-PCR) and less frequently, nested real-time PCR. In this study, we conducted a controlled comparison of two published NoV detection assays: a broadly reactive one-step real-time RT-PCR and a two-step nested real-time PCR assay. A 20% human fecal suspension containing a genogroup II human NoV was serially diluted, genome extracted, and subjected to amplification using the two assays compared via PCR Units. Additional amplicon confirmation was performed by dot blot hybridization using digoxigenin (DIG)-labeled oligonucleotide probes. Both assays displayed similar amplification standard curves/amplification efficiencies; however, the nested assay consistently detected one log10 lower virus. Dot blot hybridization improved the detection limit of the nested real-time PCR by one log10 NoV genome copies but impaired the detection limit of the one-step real-time RT-PCR by one log10 NoV genome copies. These results illustrate the complexities in designing and interpreting molecular techniques having a sufficient detection limit to detect low levels of viruses that might be anticipated in contaminated food and environmental samples.

scholarly journals Comparison of SARS-CoV2 N gene real-time RT-PCR targets and commercially available mastermixes

2020 ◽  
Author(s):  
Julianne R Brown ◽  
Denise O’Sullivan ◽  
Rui PA Pereira ◽  
Alexandra S Whale ◽  
Eloise Busby ◽  
...  
Keyword(s):  
Real Time ◽  
Lower Limit ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
N Gene ◽  
Rt Pcr ◽  
One Step ◽  
Improved Performance ◽  
Rna Transcript ◽  
Nose And Throat

ABSTRACTWe aim to test four one-step RT real-time mastermix options for use in SARS-CoV2 real-time PCR, with three primer/probe assays targeting the N gene. The lower limit of detection is determined using a SARS CoV2 N gene RNA transcript dilution series (to 1 copy/µl) and verified using 74 nose and throat swabs.The N2 assay demonstrates the most sensitive detection of SARS-Cov-2 RNA. Three of the four mastermixes performed well, with the Takara One Step PrimeScript™ III RT-PCR Kit mastermix demonstrating improved performance at the lower limit of detection.

scholarly journals A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control

2006 ◽  
Vol 6 (1) ◽  
Author(s):  
Livia Di Trani ◽  
Barbara Bedini ◽  
Isabella Donatelli ◽  
Laura Campitelli ◽  
Barbara Chiappini ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Real Time ◽  
Real Time Pcr ◽  
Positive Control ◽  
Influenza Viruses ◽  
Avian Influenza Viruses ◽  
One Step ◽  
Internal Positive Control ◽  
Mgb Probe

scholarly journals Double-Quencher Probes Improved the Detection Sensitivity of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by One-Step RT-PCR

2020 ◽  
Author(s):  
Yosuke Hirotsu ◽  
Hitoshi Mochizuki ◽  
Masao Omata
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Clinical Laboratory ◽  
Human Life ◽  
Positive Control ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Fluorescent Signal ◽  
The Usa ◽  
One Step

AbstractBackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerges in Wuhan City, Hubei Province, spreads worldwide, and threats the human life. The detection of SARS-CoV-2 is important for the prevention of the outbreak and management of patients. Real-time reverse-transcription polymerase chain reaction (RT-PCR) assay detected the virus in clinical laboratory.MethodsThis study utilized primers and single-quencher probes in accordance with the Centers for Disease Control and Prevention (CDC) in the USA and the National Institute of Infectious Diseases (NIID) in Japan. Moreover, we designed the double-quencher probes (YCH assay) according to the oligonucleotide sequence established by NIID. Using these assays, we conducted a one-step real-time RT-PCR with serial DNA positive control to assess the detection sensitivity.ResultsThe threshold cycle (Ct) value of RT-PCR was relatively low in CDC and YCH assays compared to NIID assay. Serial dilution assay showed that both CDC and YCH assays could detect a low-copy number of DNA positive control. The background fluorescent signal at the baseline was lower in YCH than that of NIID.ConclusionDouble-quencher probes decreased background fluorescent signal and improved detection sensitivity of SARS-CoV-2.

scholarly journals Positive control synthesis method for COVID-19 diagnosis by one-step real-time RT-PCR

2020 ◽  
Vol 511 ◽  
pp. 149-153
Author(s):  
Hyejin Cho ◽  
Young Hwan Jung ◽  
Hong Bum Cho ◽  
Hee-tae Kim ◽  
Kwang-sun Kim
Keyword(s):  
Real Time ◽  
Synthesis Method ◽  
Positive Control ◽  
Control Synthesis ◽  
Rt Pcr ◽  
One Step

Development of an astrovirus RT–PCR detection assay for use with conventional, real-time, and integrated cell culture/RT–PCR

2004 ◽  
Vol 50 (4) ◽  
pp. 269-278 ◽  
Author(s):  
Ann C Grimm ◽  
Jennifer L Cashdollar ◽  
Frederick P Williams ◽  
G Shay Fout
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Real Time Pcr ◽  
Environmental Water ◽  
Positive Control ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Detection Assay ◽  
Stool Samples ◽  
Pcr Method

Astrovirus is a common cause of gastroenteritis in humans that has been determined to be responsible for outbreaks of illness in several countries. Since astrovirus can be waterborne, it is important to be able to identify this virus in environmental water. We have developed and optimized a reverse transcription – polymerase chain reaction (RT–PCR) method that was able to amplify all eight astrovirus serotypes in a single reaction. In addition, a positive control construct was designed so that any inhibitors of this astrovirus assay could be detected. The assay was adapted for use in a real-time PCR assay and the sensitivity of these two methods was compared. The real-time assay was then combined with CaCo2 cell culture to produce an integrated cell culture/RT–PCR (ICC/RT–PCR) assay that was able to detect low levels of astrovirus after an incubation of 7 days or less. Also, the sensitivity of the ICC/RT–PCR assay was compared with RT–PCR alone. The methods were used to detect astrovirus in acute phase illness stool samples as well as in a water sample spiked with astrovirus.Key words: astrovirus, RT–PCR, real-time PCR, ICC/RT–PCR, environmental water.

scholarly journals Pengaruh Variasi Jumlah Tembakan Nanosecond Pulsed Electric Fields (Nspefs) Terhadap Ekspresi Gen Socs3 pada Sel Kanker Serviks Hela S3

2017 ◽  
Vol 1 (2) ◽  
pp. 45
Author(s):  
Martina Kurnia Rohmah
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Electric Fields ◽  
Real Time Pcr ◽  
Pulsed Electric Fields ◽  
Rt Pcr ◽  
Kolmogorov Smirnov ◽  
Hela S3 ◽  
Short Time ◽  
Research Show

AbstrakNanosecond Pulsed Electric Fields (NsPEFs) merupakan teknologi bioelektrik yang berkembang dari teknologi elektroporasi. NsPEFs diberikan dengan intensitas tinggi namun dalam waktu yang sangat singkat yaitu 1 - 300 nanosekon. NsPEFs terbukti memiliki sejumlah efek biologis dan telah banyak dikembangkan dalam berbagai terapi salah satunya pada terapi kanker. Pada kanker serviks, protein HPV dapat menekan sejumlah ekspresi supresor tumor salah satunya yaitu gen Socs3. Penelitian ini bertujuan untuk mengetahui pengaruh perbedaan jumlah tembakan NsPEFs pada ekspresi gen Socs3. Sel HeLa S3 dikultur pada medium α-MEM dengan serum FBS 10%. Sebesar 20 kV/cm dalam durasi 80 ns NsPEFs dipapar pada suspensi sel dalam 4 mm cuvette. Gelombang NsPEFs dideteksi oleh probe bervoltase tinggi pada Oscilloscope. NsPEFs diberikan pada 0, 5, 10, 20, 30, 40, 0, dan 60 kali tembakan. Analisis ekspresi gen dilakukan dengan dua metode yaitu kuantitatif menggunakan Real time PCR dan kualitatif dengan RT-PCR. Data kuantitatif dianalisis secara statistik menggunakan Kolmogorf-Smirnov, Anova dan HSD Tukey (p<0.05). Hasil studi ini membuktikan bahwa paparan NsPEFs berpengaruh secara signifikan pada ekspresi gen Socs3 (p=0.000). Jumlah tembakan optimal 20 dan 30 kali dapat meningkatkan ekspresi gen Socs3 berturut-turut sebanyak = 2.779 dan = 3.105 kali. Ekspresi gen Socs3 akan menurun pada tembakan di atas 30 tembakan. Kata Kunci: NsPEFs, tembakan, ekspresi, Socs3 AbstractNanosecond Pulsed Electric Fields (NsPEFs) is bioelectric that was developed by electroporation technology. NsPEFs use high intensity in short time exposure (1 – 300 nanosecond). NsPEFs have biological effect and was developed in cancer therapy. In cervical cancer, viral protein of HPV depresses some tumor suppressors like Socs3 gene. This research aims to investigate the effect of short variation in Socs3 gene expression. HeLa S3 cells were cultured in α-MEM with FBS 10%. NsPEFs as much as 20 kV/cm and 80 nano seconds was exposure over HeLa S3 cell in 4 mm cuvette. Wave of NsPEFs was detected by high voltage probe in oscilloscope. NsPEFs was exposure at 0 (control), 5, 10, 20, 30, 40, 50, and 60 shots. Socs3 gene expression was analyzed using real time PCR and RT-PCR. Quantitative data was analyzed by Kolmogorov-Smirnov, Anova, and HSD Tuker (p<0.05). This research show that NsPEFs is significantly increase Socs3 gene expression (p=0.000). The optimal shot 20 and 30 shots increase Socs3 gene expression subsequently = 2.779 and = 3.105 times. This expression decrease in higher than 30 shots of NsPEFs exposure. Keywords: NsPEFs, shot, expression, Socs3

scholarly journals One-step nested RT-PCR for COVID-19 detection: A flexible, locally developed test for SARS-CoV2 nucleic acid detection

2020 ◽  
Vol 14 (07) ◽  
pp. 679-684 ◽  
Author(s):  
Carmen Meza-Robles ◽  
Carlos E Barajas-Saucedo ◽  
Daniel Tiburcio-Jimenez ◽  
Karen A Mokay-Ramírez ◽  
Valery Melnikov ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Positive Control ◽  
Nucleic Acid Detection ◽  
Rt Pcr ◽  
Current Standard ◽  
One Step ◽  
Species Specific ◽  
Bat Coronavirus ◽  
Nucleotide Region

Introduction: Due to the coronavirus pandemic, identifying the infected individuals has become key to limiting its spread. Virus nucleic acid real-time RT-PCR testing has become the current standard diagnostic method but high demand could lead to shortages. Therefore, we propose a detection strategy using a one-step nested RT-PCR. Methodology: The nucleotide region in the ORF1ab gene that has the greatest differences between the human coronavirus and the bat coronavirus was selected. Primers were designed after that sequence. All diagnostic primers are species-specific since the 3´ end of the sequence differs from that of other species. A primer set also creates a synthetic positive control. Amplified products were seen in a 2.5% agarose gel, as well as in an SYBR Green-Based Real-Time RT-PCR. Results: Amplification was achieved for the positive control and specific regions in both techniques. Conclusions: This new technique is flexible and easy to implement. It does not require a real-time thermocycler and can be interpreted in agarose gels, as well as adapted to quantify the viral genome. It has the advantage that if the coronavirus mutates in one of the key amplification nucleotides, at least one pair can still amplify, thanks to the four diagnostic primers.

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