Binary synthetic template oligonucleotide positive control for in-house diagnostic real-time RT-PCR v2 (protocols.io.ywhfxb6)

protocols.io ◽  
2019 ◽  
Author(s):  
Ian Mackay ◽  
Judy Northill
Keyword(s):  
Real Time ◽  
Positive Control ◽  
Rt Pcr

scholarly journals Development and Testing of a Method for Detecting Lujo Virus RNA by Reverse Transcription and Real Time Polymerase Chain Reaction

2021 ◽  
pp. 110-115
Author(s):  
E. V. Naidenova ◽  
V. G. Dedkov ◽  
D. A. Agafonov ◽  
A. M. Senichkina ◽  
M. V. Safonova ◽  
...  
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Biological Samples ◽  
Control Sample ◽  
Virus Genome ◽  
Positive Control ◽  
Control Panel ◽  
Rt Pcr ◽  
Rna Detection ◽  
Virus Rna

The aim of the study was to develop and assess the efficacy of a method for Lujo virus RNA detection in clinical and biological samples using one-step real-time RT-PCR.Materials and methods. In order to select the conservative regions of the genome, we utilized the available in GenBank database Lujo virus sequences (https://www.ncbi. nlm.nih.gov/genbank) aligned in BioEdit 7.2.5 software package ( (IbisBiosciences, USA). To conduct one-round RTPCR, reverse transcriptase and TaqF-polimerase were used. Recombinant Escherichia coli strain, XL1-Blue, containing pGEM-T plasmid with inserted synthetically-generated fragment of the virus genome, was produced to make positive control sample (PCS). Constructed recombinant plasmids were used for creating RNA-containing PCS with protective protein shell of MS2-phage. Determination of specificity of the developed method was performed with the help of control panel of RNA and DNA of 23 viral strains related to 10 families; the sensitivity – the panel of biological samples artificially contaminated with PCS. Further testing was carried out at the premises of laboratory of the Russian-Guinean Center for Epidemiology and Prevention of Infectious Diseases (Kindia, Republic of Guinea) on 265 blood sera from practically healthy persons, 110 blood sera of cattle, 83 suspensions of ticks, and 165 suspensions of organs of small mammals collected in the territory of Guinea.Results and discussion. Two conservative polymerase gene fragments have been chosen as targets for Lujo virus RNA detection using RT-PCR. The combination of primers and probes has been experimentally selected, optimum composition of reaction mixture for PCR and mode of RT-PCR set-up established, as well as control samples C+, internal control, positive control sample developed. Sensitivity of the proposed method is 5·103  GE /ml, specificity – 100 %. 

scholarly journals Real-time RT-PCR detection of Citrus bark cracking viroid (CBCVd) in hops including an mRNA-based internal positive control

2020 ◽  
Vol 127 (6) ◽  
pp. 763-767
Author(s):  
Luitgardis Seigner ◽  
Marion Liebrecht ◽  
Linda Keckel ◽  
Katharina Einberger ◽  
Carolin Absmeier
Keyword(s):  
Real Time ◽  
Detection Method ◽  
Plant Protection ◽  
False Negative ◽  
Positive Control ◽  
Pcr Detection ◽  
Economic Losses ◽  
Rt Pcr ◽  
Validation Data ◽  
Internal Positive Control

Abstract Citrus bark cracking viroid (CBCVd), formerly known as pathogen in the genus Citrus and first detected in Slovenian hops in 2014, threatens hop production as it leads to important economic losses. Reduction in yield and quality and even death of the infected plants within a few years are typical observations due to CBCVd infections of hops. The viroid is easily transmitted and spreads rapidly. As it cannot be controlled by plant protection measures, avoiding its introduction into hop gardens and eradicating first centres of infection are of utmost importance. An indispensable prerequisite is a reliable detection method suitable for large-scale routine testing. In this study, the development of primers and probe for real-time RT-PCR for sensitive CBCVd detection is described. To exclude “false negative” results, a nad5 mRNA-based internal positive control was included. To our knowledge, this is the first time such a duplex real-time RT-PCR detection method for CBCVd at least in hops is described. In addition, first method validation data are presented.

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.

A Reliable Strategy for Validation of Real-Time Quantitative RT-PCR Amplicons from Linearly Amplified Human Hematopoietic Stem Cell mRNA.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4208-4208
Author(s):  
Matthew S. Nelson ◽  
Yonghong Xie ◽  
Anil C. Asrani ◽  
Pankaj Gupta
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
False Positive ◽  
Gel Electrophoresis ◽  
Positive Control ◽  
Serial Dilution ◽  
Rt Pcr ◽  
Hematopoietic Progenitors ◽  
Linear Amplification ◽  
Qrt Pcr

Abstract Real-time quantitative RT-PCR (qRT-PCR) is a powerful tool for measuring or validating gene expression. Enrichment for the most primitive hematopoietic progenitors yields small cell numbers and thus small amounts of mRNA. Linear amplification (required for gene expression analysis) amplifies the 3′ end of mRNA and imposes limitations on qRT-PCR primer design that may require the use of sub-optimal primers that can form primer dimers (PDs). Indeed, using linear amplification and qRT-PCR to assay expression of 31 genes of the bone morphogenetic protein (BMP) signaling cascade in CD34+/CD38−/lin− human umbilical cord blood (UCB) progenitors, we found that 17/31 (55%) of products were false positives. To distinguish true target amplicons from PDs, we therefore used a five-step sequential strategy to initially generate a Primer Profile for each primer set: water blank and positive control dissociation curve analysis serial dilution gel electrophoresis and product sequencing, which we describe for one gene (BMP-2) that we found is variably expressed in UCB progenitors under different culture conditions. Total RNA was obtained from 5–20 x 103 UCB progenitors cultured in conditions that induced detectable BMP-2 expression (BMP-2+) and those that did not (BMP-2−), mRNA was linearly amplified (RiboAmp) and cDNA was synthesized using Invitrogen Superscript III. qRT-PCR was performed using Invitrogen SYBR Green qPCR SuperMix on an ABI 7900 HT Sequence Detection System. (1) Though primers were designed for the 3′ end of BMP-2 using the Primer 3 website (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_slow.cgi) to identify optimal primers, an amplified product was seen in the water blank and in BMP-2− cDNA. Nevertheless, the primers also amplified the correct product in the positive control (SaOS-2 human osteosarcoma cells) and in BMP-2+ cDNA. (2) Although the genuine and the false positive products appeared similar on amplification (ΔRn vs cycle) plots, analysis of the derivative dissociation curves showed that their dissociation temperatures were distinct, being lower for the false positive PD (77°C vs 81°C). (3) Positive control and BMP-2+ cDNA yielded products with the expected one-cycle increase in cycle threshold (CT) for each 2-fold serial dilution, whereas PD product CTs did not change with serial dilution of BMP-2− cDNA. Importantly, PDs also formed at higher dilutions (≥ 1:16) of BMP-2+ cDNA, possibly because of low target mRNA abundance. (4) Agarose gel electrophoresis of the amplified products from the positive control and BMP-2+ cDNA showed the expected amplicon size of 107 bp, whereas the PD product was smaller (50 bp). (5) Finally, sequence analysis of the products (at the Univ. of Minnesota DNA Sequencing and Analysis Center) confirmed that the 107 bp product was identical with the 3′ end of human BMP-2, whereas the PD product did not yield any sequence. Similar differences were seen between genuine amplicons and PDs for several other genes examined. Thus, for each primer set, the Primer Profile provided the melting temperatures of the genuine amplicon and the PDs. For subsequent experiments, we were then able to reliably predict which product was a genuine amplicon by inspection of its melting temperature. These findings demonstrate the critical importance of using such a strategy to detect false positive qRT-PCR results due to PDs when using linear amplification of mRNA from primitive hematopoietic progenitors and other rare cell subpopulations.

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 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 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 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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