scholarly journals Development of Quantitative Real-Time Polymerase Chain Reaction for Detection of and Discrimination between Erysipelothrix Rhusiopathiae and Other Erysipelothrix Species

2009 ◽  
Vol 21 (5) ◽  
pp. 701-706 ◽  
Author(s):  
Ho To ◽  
Tomohiro Koyama ◽  
Shinya Nagai ◽  
Kotaro Tuchiya ◽  
Tetsuo Nunoya
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Limit Of Detection ◽  
Enrichment Culture ◽  
Bacterial Species ◽  
Erysipelothrix Rhusiopathiae ◽  
Chain Reaction ◽  
Tissue Samples ◽  
Practical Applications ◽  
Polymerase Chain

Quantitative real-time polymerase chain reaction (qPCR) assays were developed and validated in combination with enrichment culture for the detection and discrimination of Erysipelothrix rhusiopathiae and other Erysipelothrix species from tissue samples. The targets for SYBR green qPCR assays were the 16S ribosomal RNA gene for Erysipelothrix species and a gene involved in capsular formation for E. rhusiopathiae. The specificity of the assays was assessed with Erysipelothrix species and other related bacterial species. The limit of detection was found to be 5 colony-forming units per reaction. Amplification of DNA extracted from spleen and joint samples spiked with increasing quantities of Erysipelothrix cells was shown to be equally sensitive to DNA extracted from a pure bacterial culture. The assays were evaluated with 88 tissue samples from 3 experimentally infected pigs and 50 mice and with 36 tissue samples from 3 naturally infected pigs and 11 noninfected pigs. Results were compared with those of direct qPCR and conventional culture. The qPCR after enrichment increased the diagnostic sensitivity over that of culture and qPCR, thereby significantly reducing the total time taken for the detection of E. rhusiopathiae and other Erysipelothrix species. Therefore, this technique could be used for practical applications.

Mycoplasma pneumoniaeandChlamydophila pneumoniae: a comparative study in patients with nasal polyposis and healthy controls

2015 ◽  
Vol 129 (9) ◽  
pp. 865-869 ◽  
Author(s):  
D G Ioannidis ◽  
V A Lachanas ◽  
Z Florou ◽  
J G Bizakis ◽  
E Petinaki ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Chronic Rhinosinusitis ◽  
Nasal Polyps ◽  
Inferior Turbinate ◽  
Sinus Surgery ◽  
Control Group ◽  
Chain Reaction ◽  
Tissue Samples ◽  
Polymerase Chain

AbstractIntroduction:The role played byMycoplasma pneumoniaeandChlamydophila pneumoniaein the pathogenesis of chronic rhinosinusitis with nasal polyps has been the object of ongoing debate. We used real-time polymerase chain reaction to investigate the prevalence of both microorganisms in the nasal tissue samples of patients and controls.Methods:We extracted DNA from nasal polyp samples obtained during functional endoscopic sinus surgery and the inferior turbinate samples of controls undergoing septoplasty. We used the highly sensitive real-time polymerase chain reaction to detect the presence ofM pneumoniaeandC pneumoniaeDNA.Results:Patients with chronic rhinosinusitis with nasal polyps consisted of 62 individuals (39 men; mean age 51 years); the control group consisted of 24 individuals (13 men; mean age 45 years). All samples from both groups were negative forM pneumoniaeandC pneumoniaeDNA.Conclusion:We have demonstrated that the likelihood ofM pneumoniaeandC pneumoniaeacting as an ongoing inflammatory stimulus in chronic rhinosinusitis with nasal polyps is slim.

Detection of Naegleria fowleri, Acanthamoeba spp, and Balamuthia mandrillaris in Formalin-Fixed, Paraffin-Embedded Tissues by Real-Time Multiplex Polymerase Chain Reaction

2019 ◽  
Vol 152 (6) ◽  
pp. 799-807 ◽  
Author(s):  
Andrew P Norgan ◽  
Lynne M Sloan ◽  
Bobbi S Pritt
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Limit Of Detection ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Polymerase Chain ◽  
Formalin Fixed

Abstract Objectives Pathogenic free-living amebae (FLAs) cause skin, ocular, and central nervous system (CNS) infections with significant morbidity and mortality. Diagnosis of FLA infections by pathologic examination of tissue sections can be aided using molecular assays. This study investigated the performance characteristics of a multiplex real-time polymerase chain reaction (PCR) assay (FLA-PCR) for detection and differentiation of FLAs in clinical specimens. Methods FLA-PCR was performed on 39 human specimens comprising one cutaneous, 14 corneal, and 24 CNS formalin-fixed, paraffin-embedded (FFPE) tissues with a histopathologic diagnosis of FLA infection and four CNS FFPE tissues with inflammation but no evidence of FLAs. In addition, clinical specificity and assay limit of detection were determined. Results FLA detection sensitivities ranged from 79% to 84% in FFPE tissues. No cross-reactivity was observed. Conclusions While sensitivity is limited, FLA-PCR assay may serve as a useful adjunct for detection or confirmation of FLA infections in FFPE tissues.

scholarly journals Evaluation method for asymmetric uncertainty of quantitative polymerase chain reaction measurements of deoxyribonucleic acids with low copy number

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Unoh Ki ◽  
Takeru Suzuki ◽  
Satoshi Nakazawa ◽  
Yuuki Yonekawa ◽  
Kazuki Watanabe ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Confidence Interval ◽  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Low Copy Number ◽  
Polymerase Chain

AbstractRecently, in food safety and various other fields, qualitative and quantitative gene analysis using real-time polymerase chain reaction (PCR) method has become increasingly popular. The limit of detection (LOD) and quantifiable range for these measurements depends on the range and precision of DNA calibrators’ concentrations. Low-copy-number nucleic acid reference materials with low uncertainty produced by an inkjet system have been developed to allow for precise measurements in a low-copy-number region. However, when using a calibrator with a low copy number near one, the copy number distribution is asymmetric. Consequently, the confidence intervals of estimated copy numbers can include negative values when conventional methods of uncertainty estimation are used. A negative confidence interval is irrelevant in the context of copy number, which is always positive value or zero. Here, we propose a method to evaluate the uncertainty of real-time PCR measurements with representative values and an asymmetric 95% confidence interval. Moreover, we use the proposed method for the actual calculation of uncertainty of real-time PCR measurement results for low-copy-number DNA samples and demonstrate that the proposed method can evaluate the precision of real-time PCR measurements more appropriately in a low-copy-number region.

scholarly journals Considerations in Real-time Reverse Transcription Polymerase Chain Reaction (rRT-PCR) for the Detection of SARS-CoV-2 from Nasopharyngeal Swabs

2021 ◽  
pp. 68-78
Author(s):  
Priyadharshini Sekar ◽  
Godfred Antony Menezes ◽  
Pooja Shivappa ◽  
Biji Thomas George ◽  
Ashfaque Hossain
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reverse Transcription ◽  
Limit Of Detection ◽  
Disease Diagnosis ◽  
Antigen Detection ◽  
Disease Spread ◽  
Chain Reaction ◽  
The World ◽  
Polymerase Chain

Coronavirus Disease 2019 (COVID-19) was first reported in December 2019, in the City of Wuhan, China. Within the span of a few weeks, the disease had spread to other regions of China and eventually to different parts of the world. COVID 19 has affected 221 countries and territories around the world, with a total of 121,290,697 positive cases and 2,682,554 deaths as on March 17, 2021. Accurate disease diagnosis (for the SARS-Cov-2 virus and variants), coupled to patient isolation are currently critical strategies in restricting disease spread. Due to lack of time during this pandemic the diagnostics assays were not adequately validated. Infected individuals at times could potentially be missed by real-time reverse transcription polymerase chain reaction (rRT-PCR) for SARS-CoV-2 tests due to incorrect/inefficient sampling procedure, low limit of detection and epidemiology of the virus. rRT-PCR test results should be interpreted in conjunction with clinical examination and Computed Tomography (CT), particularly in suspected symptomatic individuals or those with epidemiological history of contact with known COVID-19 cases. Considering the above-mentioned constraints, the current scenario demands rapid and point-of-care tests for detection of SARS-CoV-2 in remote locations. To date, there is no reliable commercially available antigen detection kit. The infected subjects reveal low levels of antibodies against SARS-CoV-2 through the early period of infection. In addition, techniques such as, Digital RT-PCR technology and isothermal RNA amplification with electrochemical biosensors are some of the new technologies currently being developed to provide sensitive and specific SARS-Cov-2 antigen detection. The newly reported variant, SARS-CoV-2 VUI 202012/01 may not influence diagnostic outcomes as worldwide most PCR assays use two or more (including RdRp/ E/ N) reliable gene targets, besides S gene.

scholarly journals Quantitative Multiplex Real-Time Reverse Transcriptase–Polymerase Chain Reaction with Fluorescent Probe Detection of Killer Immunoglobulin-Like Receptors, KIR2DL4/3DL3

Diagnostics ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 588
Author(s):  
Wipaporn Wongfieng ◽  
Rungtiwa Nutalai ◽  
Amonrat Jumnainsong ◽  
Chanvit Leelayuwat
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Limit Of Detection ◽  
Expression Patterns ◽  
Multiplex Assay ◽  
Clinical Samples ◽  
Lower Sensitivity ◽  
Chain Reaction ◽  
Kir Genes ◽  
Polymerase Chain

(1) Background: KIR2DL4/KIR3DL3 are the framework genes present in all KIR haplotypes, with unique expression patterns being present only in women and CD56bright NK cells. KIR genes have a high degree of DNA sequence identity. Consequently, they are one of the most challenging genes for molecular detection—especially regarding expressions; (2) Methods: We developed an effective method to determine KIR3DL3/KIR2DL4 expressions based on a multiplex quantitative real-time Reverse transcription polymerase chain reaction (qRT-PCR )with fluorescent probes using NK92; (3) Results: Standardizations of the singleplex KIR2DL4 and KIR3DL3 were performed to evaluate the sensitivity and specificity for further development of the multiplex assay. The limit of detection was at 500 copies each. There was cross-amplification with the presence of related KIR genes at a level of 5 × 107 copies. This is not biologically significant because this high level of KIR expression has not been found in clinical samples. The multiplex assay was reproducible equivalent to its singleplex (KIR2DL4; R2 = 0.995, KIR3DL3; R2 = 0.996, but lower sensitivity of 103 copies). Furthermore, the validation of the developed method on samples of blood donors showed high sensitivity (100%) and specificity (99.9%); (4) Conclusions: The developed method is reliable and highly specific suitable for evaluation of the KIR2DL4/3DL3 mRNA expressions in further applications.

scholarly journals Development of a One-Step Real-Time Polymerase Chain Reaction Assay for Diagnosis of Phytophthora ramorum

2006 ◽  
Vol 96 (9) ◽  
pp. 975-981 ◽  
Author(s):  
Kelvin J. D. Hughes ◽  
Jennifer A. Tomlinson ◽  
Ruth L. Griffin ◽  
Neil Boonham ◽  
Alan J. Inman ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Host Plant ◽  
Real Time ◽  
Plant Material ◽  
Limit Of Detection ◽  
Phytophthora Ramorum ◽  
Chain Reaction ◽  
Simple Method ◽  
Isolation Technique ◽  
Polymerase Chain

Phytophthora ramorum is a recently described pathogen causing bleeding cankers, dieback, and leaf blight on trees and shrubs in parts of Europe and North America, where the disease is commonly known as sudden oak death. This article describes the development of a single-round real-time polymerase chain reaction (PCR) assay based on TaqMan chemistry, designed within the internal transcribed spacer 1 region of the nuclear ribosomal (nr)RNA gene for detection of P. ramorum in plant material. Unlike previously described methods for the molecular detection of P. ramorum, this assay involves no post amplification steps or multiple rounds of PCR. The assay was found to have a limit of detection of 10 pg of P. ramorum DNA, and could detect P. ramorum in plant material containing 1% infected material by weight within 36 cycles of PCR. The assay also was used to test DNA from 28 other Phytophthora spp. to establish its specificity for P. ramorum. A quick and simple method was used to extract DNA directly from host plant material, and detection of P. ramorum was carried out in multiplex with an assay for a gene from the host plant in order to demonstrate whether amplifiable DNA had been extracted. Amplifiable DNA was extracted from 84.4% of samples, as demonstrated by amplification of host plant DNA. The real-time protocol was used to test 320 plant samples (from 19 different plant species) from which DNA extraction had been successful, and was shown to give results comparable with a traditional isolation technique for diagnosis of P. ramorum in plant material from common U.K. hosts.

scholarly journals Investigation of SP1 and SP3 Expressions in Colorectal Cancer Carcinogenesis

2021 ◽  
Vol 21 (03) ◽  
Author(s):  
Turkan Gurer
Keyword(s):  
Colorectal Cancer ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Pathological Features ◽  
Chain Reaction ◽  
Tissue Samples ◽  
High Positive Correlation ◽  
Pathological Characteristics ◽  
Significant Difference ◽  
Polymerase Chain

ABSTRACT The study aimed to determine the expressions of SP1 and SP3 and the clinic pathological characteristics of patients, and the correlation between the expressions of SP1 and SP3 in colorectal cancer (CRC). In this study, tumour and adjacent non-tumour tissue samples were obtained from 41 individuals with CRC. SP1 and SP3 expressions were performed using Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR). According to the results of our study, there was no statistically significant difference in SP1 and SP3 expression levels between tumour tissues and non-tumour tissues (p>0.05), as well as no association with clinic pathological features of patients. In addition, a high positive correlation was found between the expressions of SP1 and SP3 genes in CRC (p=0.01). Consequently, it can be said that there is a correlation between SP1 and SP3 expressions, but SP1 and SP3 expressions are not related to CRC carcinogenesis.

scholarly journals Comparative evaluation of a competitive polymerase chain reaction (PCR) and a SYBR Green–based real-time PCR to quantify Porcine circovirus-2 DNA in swine tissue samples

2011 ◽  
Vol 23 (6) ◽  
pp. 1160-1167 ◽  
Author(s):  
Diogenes Dezen ◽  
Franciscus A.M. Rijsewijk ◽  
Thais F. Teixeira ◽  
Carine L. Holz ◽  
Ana P. Varela ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Manifestations ◽  
Sybr Green ◽  
Porcine Circovirus ◽  
Chain Reaction ◽  
Tissue Samples ◽  
Porcine Circovirus 2 ◽  
Polymerase Chain

Porcine circovirus-2 (PCV-2) is considered the major etiological agent of post-weaning multisystemic wasting syndrome (PMWS) in pigs. The clinical manifestations of the disease are correlated with moderate to high amounts of PCV-2 DNA in biological samples of affected pigs. A threshold of 107 DNA copies/ml is suggested as the trigger factor for symptoms. A comparative study was conducted to determine which quantitative method would be more suitable to estimate the PCV-2 DNA load. Two polymerase chain reaction (PCR) assays were developed: a competitive PCR (cPCR) and a SYBR Green–based real-time PCR. The assays were compared for their capacity to detect PCV-2 in DNA samples extracted from liver, lung, spleen, mesenteric lymph nodes, and kidney of PMWS-affected ( n = 23) or non–PMWS-affected pigs ( n = 9). Both assays could successfully quantify PCV-2 DNA in all tissue samples and were able to detect significant differences between the numbers of PCV-2 DNA copies found in tissues of PMWS-affected and non–PMWS-affected pigs (≥102.5). The highest mean viral loads were detected by the SYBR Green real-time PCR, up to 107.0±1.5 copies/100 ng of total DNA sample, while the cPCR detected up to 104.8±1.5. A mean difference of 101.8 was found between the amounts of PCV-2 DNA detected, using the SYBR Green real-time PCR and the cPCR, suggesting that the viral load threshold for PMWS should be determined for each particular assay.

A report of swine erysipelas infection in an organised farm in Kerala

2019 ◽  
Author(s):  
S. Sankar ◽  
P.S. Reshma ◽  
N. Sarika ◽  
M.R. Roshin ◽  
R. Niranjana ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Skin Infections ◽  
Post Mortem ◽  
Erysipelothrix Rhusiopathiae ◽  
Chain Reaction ◽  
Tissue Samples ◽  
Biochemical Characteristics ◽  
Source Of Infection ◽  
Polymerase Chain ◽  
Swine Erysipelas

Erysipelothrix rhusiopathiae is an established animal pathogen causing erysipelas in animals and occasionally it causes zoonotic skin infections in humans, known as erysipeloid. The present study was aimed to investigate the cause of sudden mortality in a batch of gilts in an organised farm in Thrissur district of Kerala. The heart swabs and tissue samples (spleen, liver, lungs, and heart) collected during post-mortem examination yielded growth of small Gram positive, non-capsulated, spore forming pleomorphic bacilli. Based on cultural, morphological and biochemical characteristics, these isolates were identified as E. rhusiopathiae. Furthermore, the isolates were subjected to Erysipelothrix specific 16S rRNA based polymerase chain reaction. The isolates were sequenced for further confirmation. The isolates were confirmed as E. rhusiopathiae by phenotypic and genotypic characterisation. Timely diagnosis of the disease helped to identify the possible source of infection. This study highlights the importance of timely identification of E. rhusiopathiae infection in an outbreak; thereby adequate strategies can be implemented to control the infection.

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