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

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.

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

10.1101/2020.11.04.20209635 ◽

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.

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Development of an efficient one-step real-time reverse transcription polymerase chain reaction method for severe acute respiratory syndrome-coronavirus-2 detection

PLoS ONE ◽

10.1371/journal.pone.0252789 ◽

2021 ◽

Vol 16 (6) ◽

pp. e0252789

Author(s):

Yukiko Nakura ◽

Heng Ning Wu ◽

Yuya Okamoto ◽

Muneyuki Takeuchi ◽

Koichiro Suzuki ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Reverse Transcriptase ◽

Real Time ◽

Dna Polymerase ◽

Internal Control ◽

Leukemia Virus ◽

Rt Pcr ◽

Thermotoga Petrophila ◽

One Step ◽

Thermostable Dna Polymerase

The general methods to detect the RNA of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in clinical diagnostic testing involve reverse transcriptases and thermostable DNA polymerases. In this study, we compared the detection of SARS-CoV-2 by a one-step real-time RT-PCR method using a heat-resistant reverse transcriptase variant MM4 from Moloney murine leukemia virus, two thermostable DNA polymerase variants with reverse transcriptase activity from Thermotoga petrophila K4 and Thermococcus kodakarensis KOD1, or a wild-type DNA polymerase from Thermus thermophilus M1. The highest performance was achieved by combining MM4 with the thermostable DNA polymerase from T. thermophilus M1. These enzymes efficiently amplified specific RNA using uracil-DNA glycosylase (UNG) to remove contamination and human RNase P RNA amplification as an internal control. The standard curve was obtained from 5 to 105 copies of synthetic RNA. The one-step real-time RT-PCR method’s sensitivity and specificity were 99.44% and 100%, respectively (n = 213), compared to those of a commercially available diagnostic kit. Therefore, our method will be useful for the accurate detection and quantification of SARS-CoV-2.

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

10.1101/2020.08.06.20168294 ◽

2020 ◽

Cited By ~ 1

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.

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Rapid Differential Detection of H1 and H3 Subtype Swine Influenza Viruses by a TaqMan-MGB Based Duplex One-Step Real Time RT-PCR Assay

10.21203/rs.3.rs-306178/v1 ◽

2021 ◽

Author(s):

Kaibiao Chen ◽

Ming Kong ◽

Jiao Liu ◽

Jun Jiao ◽

Zixiong Zeng ◽

...

Keyword(s):

Real Time ◽

Swine Influenza ◽

Detection Sensitivity ◽

Differential Detection ◽

Rt Pcr ◽

Pcr Assay ◽

H3n2 Subtype ◽

One Step ◽

H3 Subtype ◽

Probe Set

Abstract Swine influenza is not only an economically important respiratory disease in swine, but also constantly poses a threat to human health. Hence, developing a rapid, sensitive and efficient detection method of swine influenza virus (SIV) is highly essential. By aligning the HA gene sequences of SIV circulating in China in recent 10 years, a H1 primer-probe set targeting both Eurasian avian-like H1N1 (EA H1N1) and Pandemic 2009 H1N1 (Pdm09 H1N1) lineages plus a H3 prime-probe set targeting the prevalent human-like H3N2 (HL H3N2) subtype were designed, respectively. Further, a TaqMan-MGB based duplex one-step real time RT-PCR (RRT-PCR) assay was established and evaluated. The duplex RRT-PCR possessed the detection limit of 5 copies/μL HA plasmid for each of the EA H1N1, Pdm09 H1N1 and HL H3N2 subtype SIVs, and matched an overall detection sensitivity of 100% and specificity of 91.67% with traditional virus isolation through chicken embryo inoculation using experimentally infected mice lung samples. Besides, the method showed high repeatability both within-run and between-runs, and no cross-reactivity against some commonly circulated porcine viruses in China. Furthermore, the duplex RRT-PCR method revealed a relatively higher prevalent rate of H1 than H3 subtype SIV in 166 nasal swabs from pigs in some slaughterhouse during October ~ December, 2019. This developed assay could be very helpful for rapid differential detection and routine surveillance of EA H1N1, Pdm09 H1N1 and HL H3N2 subtype SIVs in China.

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

Clinica Chimica Acta ◽

10.1016/j.cca.2020.10.001 ◽

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

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

The Journal of Infection in Developing Countries ◽

10.3855/jidc.12726 ◽

2020 ◽

Vol 14 (07) ◽

pp. 679-684 ◽

Cited By ~ 2

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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