scholarly journals Optimisation of rapid untargeted nanopore DNA virus metagenomics using cell cultures and calves experimentally infected with bovine herpes virus-1

2021 ◽  
Author(s):  
Gaelle Esnault ◽  
Bernadette Earley ◽  
Paul Cormican ◽  
Sinead M. Waters ◽  
Ken Lemon ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Cell Cultures ◽  
Pcr Amplification ◽  
Acid Extraction ◽  
Chain Reaction ◽  
Dna Viruses ◽  
Nasal Swabs ◽  
Polymerase Chain ◽  
Bovine Herpes Virus 1 ◽  
Foetal Lung

Abstract Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality in cattle in Ireland, and internationally. This disease is caused by many well-known, and an ever-increasing number of newly associated viruses and bacteria. Consequently, diagnosis of BRD pathogens by targeted real-time polymerase chain reaction (qPCR) diagnostics is too expensive and slow to enable a same-day response that is targeted at the causative pathogen(s). To address this, we developed a same-day, sample to result, untargeted metagenomic MinION sequencing protocol for the identification of DNA viruses associated with BRD from nasal swabs. The procedure comprises non-viral nucleic acid depletion, nucleic acid extraction, rapid transposase-based tagmentation with barcoded adapters, non-biased PCR amplification of tagmented nucleic acid, sequencing on a MinION device, then rapid analysis of resulting sequences on cloud-based software EPI2ME WIMP. The protocol was developed using BoHV1-infected foetal lung cell cultures where we achieved 96% enrichment of the BoHV-1 sequence. Subsequently, the protocol was successfully applied to untargeted detection of BoHV-1 in nasal swabs from calves experimentally challenged with BoHV-1.

scholarly journals Use of Touch-Down Polymerase Chain Reaction to Enhance the Sensitivity of Mycobacterium Bovis Detection

2005 ◽  
Vol 17 (3) ◽  
pp. 232-238 ◽  
Author(s):  
Martín J. Zumárraga ◽  
Virginia Meikle ◽  
Amelia Bernardelli ◽  
Alejandro Abdala ◽  
Hector Tarabla ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Mycobacterium Bovis ◽  
Pcr Amplification ◽  
Sensitive Detection ◽  
Chain Reaction ◽  
Single Tube ◽  
Nasal Swabs ◽  
Diagnostic Samples ◽  
Polymerase Chain ◽  
Duplication Time

The confirmatory diagnosis of Mycobacterium bovis ( M. bovis) in animal samples is carried out by culture in Stonebrink media. However, culture is very slow because of the extremely long duplication time of the bacillus and difficult because of the scarcity of bacilli in diagnostic samples. This study describes the development of a single-tube touch-down polymerase chain reaction (PCR) protocol for the detection of M. bovis using primers that target the IS 6110 element. Spiked water and milk as well as routine diagnostic samples (milk and nasal swabs) from M. bovis–positive cattle were tested. This protocol allows the rapid and sensitive detection of M. bovis in bovine samples by enhancing the sensitivity of standard PCR amplification.

scholarly journals Optimization of Real-time Reverse Transcriptase Polymerase Chain Reaction for Detection of Dengue Virus

2020 ◽  
pp. 1-7
Author(s):  
Kaunara A. Azizi ◽  
Arnold J. Ndaro ◽  
Athanasia Maro ◽  
Adonira Saro ◽  
Reginald A. Kavishe
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Dengue Virus ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Reliable Technique ◽  
Polymerase Chain

Aims: This study was set to optimize conditions for real time reverse transcriptase polymerase chain reaction (RT-PCR) for detection of dengue virus by using rapid and simple nucleic acid extraction method. Methodology: One step and two step real time RT-PCR were evaluated in different PCR thermocyclers. Extraction of viral RNA was done by using a simple boom method. Results: The real time RT-PCR technique was successfully optimized using simple and rapid method for purification of nucleic acid, ‘boom method’. The technique works better when performed in a two-step procedure and can works well with all range of real time PCR machines. The optimized real time RT-PCR used in the present study is a valuable and reliable technique for routine diagnosis of dengue. Further investigation on the cost effectiveness in adopting this technique for routine screening and monitoring of the dengue infection should be done.

Application of Magnetic/Magnetic Fluorescent Microsphere Preparation in Genes Food Testing

2014 ◽  
Vol 644-650 ◽  
pp. 5356-5359
Author(s):  
Yong Hui Liu
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Extraction Method ◽  
Genetically Modified ◽  
High Price ◽  
Ammonium Bromide ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Chain Reaction ◽  
Polymerase Chain

Polymerase chain reaction (PCR) and fluorescence quantitative polymerase chain reaction (qRT - PCR) is a major means of detection of genetically modified food, whose key is to extract high quality nucleic acid in quick and high flux. The main nucleic acid extraction method is hexadecyl trimethyl ammonium bromide (CTAB) and sodium dodecyl sulfate (SDS) and kits. Because operation of CTAB and SDS method are cumbersome, require the use of organic solvent and centrifugal unceasingly, thus can not meet the requirement of the rapid development of high throughput; and because of the high price of kit method, its application in the daily inspection is also limited. At the same time, the development trend of genetically modified detection based on nucleic acid level is multiple tests; the key is the preparation of different kinds of fluorescent microspheres and specific probe of microspheres. To solve above problems, this paper put forward nucleic acid extraction method on the basis of the magnetic separation technology.

Design and Implementation of Polymerase Chain Reaction Device for Aptamers Selection of Tumor Cells

2020 ◽  
Vol 20 (3) ◽  
pp. 1332-1340
Author(s):  
Chao Wang ◽  
Fan Meng ◽  
Yanping Huang ◽  
Nongyue He ◽  
Zhu Chen
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Tumor Cells ◽  
Pcr Amplification ◽  
Chain Reaction ◽  
Control Module ◽  
Aptamer Selection ◽  
Nucleic Acid Aptamers ◽  
Polymerase Chain ◽  
Selection Of

Nucleic acid aptamers are a kind of one-dimensional biological nanomaterials and have found many applications. This paper designed and implemented a polymerase chain reaction (PCR) amplification device with a reaction volume of 500 μL, which can be used for the amplification of nucleic acid aptamers of tumor cells in the aptamer selection. This device mainly includes a control module, a temperature measuring module, a PCR amplification tube, a metal tank module, a liquid crystal display (LCD) and operation module and a cooling module. The new PCR amplification chamber is matched with the designed metal tank to ensure the temperature uniformity of the PCR amplification solution. The control module based on the STM32F103RCT6 manages the workflow of the entire device. The PCR amplification chamber and PT100 sensors on the metal tank formed a closed-loop feedback system, and the incremental proportional-integral-derivative (PID) algorithm was used to achieve the precise temperature control. In addition, we introduced the Smith predictive compensation algorithm to solve the temperature hysteresis problem of the PCR amplification chamber. The experimental results showed that the PCR device can meet the requirements for the nucleic acid aptamer selection of tumor cells. The device can also be used in other experiments with large-volume PCR amplification.

scholarly journals Amplification of human beta-actin gene by the reverse transcriptase-polymerase chain reaction: implications for assessment of RNA from formalin-fixed, paraffin-embedded material.

1996 ◽  
Vol 44 (10) ◽  
pp. 1205-1207 ◽  
Author(s):  
A Dakhama ◽  
V Macek ◽  
J C Hogg ◽  
R G Hegele
Keyword(s):  
Polymerase Chain Reaction ◽  
Pcr Amplification ◽  
Housekeeping Gene ◽  
Chain Reaction ◽  
Viral Genomes ◽  
Negative Results ◽  
Beta Actin ◽  
Target Nucleic Acid ◽  
Formalin Fixed Paraffin Embedded ◽  
Polymerase Chain

The polymerase chain reaction (PCR) is a powerful method that allows enzymatic amplification of rate target nucleic acid sequences. It has been applied to the amplification of viral genomes from paraffin-embedded pathology specimens. However, interpretation of negative results requires amplification of a housekeeping gene such as beta-actin. In the present study we used specific oligonucleotide primers previously designed to amplify both the genomic DNA and the mRNA transcript from paraffin-embedded tissue. These products have predicted sizes of 250 BP and 154 BP, respectively, but our results showed that PCR amplification only (without reverse transcription) unexpectedly generated the 154-BP product. Further investigation of the nature of this product demonstrated that it originated from the amplification of DNA, not RNA. We conclude that the 154-BP product generated by these primers cannot be exclusively considered as beta-actin RNA product and should not be used to assess successful extraction of RNA, to ascertain its integrity, or to normalize for the total amount of RNA assayed by RT-PCR from paraffin-embedded tissue.

scholarly journals A Self-Contained Portable Polymerase-Chain-Reaction System Integrated with Electromagnetic Mini-Actuators for Bi-Directional Fluid Transport

2011 ◽  
Vol 27 (3) ◽  
pp. 357-364
Author(s):  
B. T. Chia ◽  
S.-A. Yang ◽  
M.-Y. Cheng ◽  
C.-W. Lin ◽  
Y.-J. Yang
Keyword(s):  
Polymerase Chain Reaction ◽  
Fluid Transport ◽  
Point Of Care ◽  
Reaction System ◽  
Thermal Characteristics ◽  
Pcr Amplification ◽  
Chain Reaction ◽  
Rapid Temperature ◽  
Small Footprint ◽  
Polymerase Chain

ABSTRACTIn this paper, the development of a portable polymerase chain reaction (PCR) device is presented. Integrating electromagnetic mini-actuators for bi-directional fluid transport, the proposed device, whose dimension is 67mm × 66mm × 25mm, can be fully operated with a 5V DC voltage. The device consists of four major parts: A disposable channel chip in which PCR mixture is manipulated and reacted, a heater chip which generates different temperature zones for PCR reaction, a linear actuator array for pumping PCR mixture, and a circuit module for controlling and driving the system. The advantages of the device include the rapid temperature responses associated with continuous-flow-type PCR devices, as well as the programmable thermal cycling associated with chamber-type PCR devices. The thermal characteristics are measured and discussed. PCR amplification is successfully performed for the 122 bp segment of MCF-7/adr cell line. Due to its small footprint, this self-contained system potentially can be employed for point-of-care (POC) applications.

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