scholarly journals High-Volume Extraction of Nucleic Acids by Magnetic Bead Technology for Ultrasensitive Detection of Bacteria in Blood Components

2007 ◽  
Vol 53 (1) ◽  
pp. 104-110 ◽  
Author(s):  
Melanie Störmer ◽  
Knut Kleesiek ◽  
Jens Dreier
Keyword(s):  
Detection Limit ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Real Time ◽  
High Volume ◽  
Magnetic Bead ◽  
Locked Nucleic Acid ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Probe System

Abstract Background: Nucleic acid isolation, the most technically demanding and laborious procedure performed in molecular diagnostics, harbors the potential for improvements in automation. A recent development is the use of magnetic beads covered with nucleic acid–binding matrices. We adapted this technology with a broad-range 23S rRNA real-time reverse transcription (RT)-PCR assay for fast and sensitive detection of bacterial contamination of blood products. Methods: We investigated different protocols for an automated high-volume extraction method based on magnetic-separation technology for the extraction of bacterial nucleic acids from platelet concentrates (PCs). We added 2 model bacteria, Staphylococcus epidermidis and Escherichia coli, to a single pool of apheresis-derived, single-donor platelets and assayed the PCs by real-time RT-PCR analysis with an improved primer–probe system and locked nucleic acid technology. Coamplification of human β2-microglobulin mRNA served as an internal control (IC). We used probit analysis to calculate the minimum concentration of bacteria that would be detected with 95% confidence. Results: For automated magnetic bead–based extraction technology with the real-time RT-PCR, the 95% detection limit was 29 × 103 colony-forming units (CFU)/L for S. epidermidis and 22 × 103 CFU/L for E. coli. No false-positive results occurred, either due to nucleic acid contamination of reagents or externally during testing of 1030 PCs. Conclusions: High-volume nucleic acid extraction improved the detection limit of the assay. The improvement of the primer–probe system and the integration of an IC make the RT-PCR assay appropriate for bacteria screening of platelets.

scholarly journals Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis

PLoS ONE ◽  
2015 ◽  
Vol 10 (11) ◽  
pp. e0143444 ◽  
Author(s):  
Yong Zhao ◽  
Guilian Li ◽  
Chongyun Sun ◽  
Chao Li ◽  
Xiaochen Wang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Locked Nucleic Acid ◽  
Pcr Assay ◽  
Locked Nucleic Acid Probe

scholarly journals Development of a Conventional RT-PCR Assay for Rapid Detection of Porcine Deltacoronavirus with the Same Detection Limit as a SYBR Green-Based Real-Time RT-PCR Assay

2018 ◽  
Vol 2018 ◽  
pp. 1-7
Author(s):  
Lei Ma ◽  
Fanwen Zeng ◽  
Bihong Huang ◽  
Feng Cong ◽  
Ren Huang ◽  
...  
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Limit Of Detection ◽  
Sybr Green ◽  
Watery Diarrhea ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Rna Molecules ◽  
Positive Rate

Porcine deltacoronavirus (PDCoV) is a newly discovered coronavirus, which belongs to the family Coronaviridae. It causes watery diarrhea, vomiting, and dehydration in newborn piglets. A sensitive RT-PCR method is urgently required to detect PDCoV infection. In this study, we developed and evaluated a conventional RT-PCR assay and a SYBR green-based real-time RT-PCR assay that targeted the PDCoV n gene. Both assays are specific and have the same limit of detection at 2 × 101 copies of RNA molecules per reaction. Eighty-four clinical samples were subjected to both conventional RT-PCR and real-time RT-PCR, and the same positive rate (41.7%) was achieved, which was much higher than the positive rate (26.2%) using a previously described one-step RT-PCR technique. In summary, a conventional RT-PCR technique was successfully established for the detection of PDCoV with the same detection limit as a SYBR green-based real-time RT-PCR assay.

scholarly journals Using the LN34 Pan-Lyssavirus Real-Time RT-PCR Assay for Rabies Diagnosis and Rapid Genetic Typing from Formalin-Fixed Human Brain Tissue

Viruses ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 120 ◽  
Author(s):  
Rene Edgar Condori ◽  
Michael Niezgoda ◽  
Griselda Lopez ◽  
Carmen Acosta Matos ◽  
Elinna Diaz Mateo ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Dominican Republic ◽  
Real Time ◽  
Brain Tissue ◽  
Border Region ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Fixed Tissue ◽  
Genetic Typing ◽  
Formalin Fixed

Human rabies post mortem diagnostic samples are often preserved in formalin. While immunohistochemistry (IHC) has been routinely used for rabies antigen detection in formalin-fixed tissue, the formalin fixation process causes nucleic acid fragmentation that may affect PCR amplification. This study reports the diagnosis of rabies in an individual from the Dominican Republic using both IHC and the LN34 pan-lyssavirus real-time RT-PCR assay on formalin-fixed brain tissue. The LN34 assay generates a 165 bp amplicon and demonstrated higher sensitivity than traditional PCR. Multiple efforts to amplify nucleic acid fragments larger than 300 bp using conventional PCR were unsuccessful, probably due to RNA fragmentation. Sequences generated from the LN34 amplicon linked the case to the rabies virus (RABV) strain circulating in the Ouest Department of Haiti to the border region between Haiti and the Dominican Republic. Direct sequencing of the LN34 amplicon allowed rapid and low-cost rabies genetic typing.

scholarly journals A one-step real-time RT-PCR assay for simultaneous typing of SARS-CoV-2 mutations associated with the E484K and N501Y spike protein amino-acid substitutions

2021 ◽  
Author(s):  
Serafeim C. Chaintoutis ◽  
Taxiarchis Chassalevris ◽  
George Tsiolas ◽  
Sofia Balaska ◽  
Ioannis Vlatakis ◽  
...  
Keyword(s):  
Amino Acid ◽  
Real Time ◽  
Immune Escape ◽  
Rapid Identification ◽  
Locked Nucleic Acid ◽  
Amino Acid Substitutions ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Protein Amino Acid ◽  
One Step

The emergence of SARS-CoV-2 mutations resulting in the S protein amino-acid substitutions N501Y and E484K, which have been associated with enhanced transmissibility and immune escape, respectively, necessitates immediate actions, for which their rapid identification is crucial. For the simultaneous typing of both of these mutations of concern (MOCs), a one-step real-time RT-PCR assay employing four locked nucleic acid (LNA) modified TaqMan probes was developed. The assay is highly sensitive with a LOD of 117 copies/reaction, amplification efficiencies >94% and a linear range of over 5 log10 copies/reaction. Validation of the assay using known SARS-CoV-2-positive and negative samples from human and animals revealed its ability to correctly identify wild type strains, and strains possessing either one or both targeted amino-acid substitutions, thus comprising a useful pre-screening tool for rapid MOC identification. The basic principles of the methodology for the development of the assay are explained in order to facilitate the rapid design of similar assays able to detect emerging MOCs.

Promising Nucleic Acid Lateral Flow Assay Plus PCR for Shiga Toxin–Producing Escherichia coli

2015 ◽  
Vol 78 (8) ◽  
pp. 1560-1568 ◽  
Author(s):  
YOSHITAKA TERAO ◽  
KANA TAKESHITA ◽  
YASUTAKA NISHIYAMA ◽  
NAOKI MORISHITA ◽  
TAKASHI MATSUMOTO ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Detection Limit ◽  
Nucleic Acid ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Pcr Assay ◽  
E Coli ◽  
Lower Detection Limit ◽  
Lower Detection

Shiga toxin (Stx)–producing Escherichia coli (STEC) is a frequent cause of foodborne infections, and methods for rapid and reliable detection of STEC are needed. A nucleic acid lateral flow assay (NALFA) plus PCR was evaluated for detecting STEC after enrichment. When cell suspensions of 45 STEC strains, 14 non-STEC strains, and 13 non–E. coli strains were tested with the NALFA plus PCR, all of the STEC strains yielded positive results, and all of the non-STEC and non–E. coli strains yielded negative results. The lower detection limit for the STEC strains ranged from 0.1 to 1 pg of genomic DNA (about 20 to 200 CFU) per test, and the NALFA plus PCR was able to detect Stx1- and Stx2-producing E. coli strains with similar sensitivities. The ability of the NALFA plus PCR to detect STEC in enrichment cultures of radish sprouts, tomato, raw ground beef, and beef liver inoculated with 10-fold serially diluted STEC cultures was comparable to that of a real-time PCR assay (at a level of 100 to 100,000 CFU/ml in enrichment culture). The bacterial inoculation test in raw ground beef revealed that the lower detection limit of the NALFA plus PCR was also comparable to that obtained with a real-time PCR assay that followed the U.S. Department of Agriculture guidelines. Although further evaluation is required, these results suggest that the NALFA plus PCR is a specific and sensitive method for detecting STEC in a food manufacturing plant.

scholarly journals New Real-Time PCR Assay Using Locked Nucleic Acid Probes To Assess Prevalence of ParC Mutations in Fluoroquinolone-Susceptible Streptococcus pneumoniae Isolates from France

2006 ◽  
Vol 50 (4) ◽  
pp. 1594-1598 ◽  
Author(s):  
Jean-Winoc Decousser ◽  
Imen Methlouthi ◽  
Patrick Pina ◽  
Anne Collignon ◽  
Pierre Allouch
Keyword(s):  
Streptococcus Pneumoniae ◽  
Nucleic Acid ◽  
High Risk ◽  
Real Time ◽  
Real Time Pcr ◽  
Locked Nucleic Acid ◽  
Pcr Assay ◽  
Nucleic Acid Probes ◽  
National Surveys ◽  
Risk Patients

ABSTRACT A real-time PCR assay with locked nucleic acid probes was developed to screen mutations at codons 79 and 83 of the Streptococcus pneumoniae parC gene. Only silent mutations were detected among 236 French invasive fluoroquinolone-susceptible strains. This test could be useful for some high-risk patients or in national surveys.

scholarly journals Real-Time Detection of the Mutation Responsible for Progressive Rod-Cone Degeneration in Labrador Retriever Dogs Using Locked Nucleic Acid TaqMan Probes

2009 ◽  
Vol 21 (5) ◽  
pp. 689-692 ◽  
Author(s):  
Fabio Gentilini ◽  
Gian Luca Rovesti ◽  
Maria Elena Turba
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Late Onset ◽  
Labrador Retriever ◽  
Locked Nucleic Acid ◽  
Sensitivity Threshold ◽  
Ophthalmological Examination ◽  
Pcr Assay ◽  
Labrador Retrievers

Progressive rod-cone degeneration ( prcd) is a late onset, autosomal recessive, inherited disease in dogs caused by a G > A substitution in the PRCD locus, which has been reported in more than 18 breeds, including Labrador Retriever dogs. In this study, a real-time polymerase chain reaction (PCR) assay, exploiting the features of locked nucleic acid (LNA) fluorescent-labeled probes, was developed to genotype the sequence variants responsible for the disease. Two Labrador Retrievers were diagnosed with prcd by ophthalmological examination performed by a panelist of the Italian hereditary eye disease control program. The 2 dogs, as well as 8 related and 14 unrelated Labrador Retrievers, were genotyped with both direct sequencing of the disease locus and real-time LNA TaqMan PCR assay. Even though the region surrounding the mutation was predicted to be highly structured, making probe annealing difficult, the real-time PCR assay allowed researchers to correctly genotype the dogs in all cases with a sensitivity threshold of 4 ng/reaction of genomic DNA. A real-time PCR assay will allow a high-throughput analysis of a larger cohort of dogs, thereby enabling researchers to investigate the prevalence of the mutated allele in the affected breeds.

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