Whole blood Nested PCR and Real-time PCR amplification ofTalaromyces marneffeispecific DNA for diagnosis

Here we report a new real-time PCR assay using SYBR Green which provides higher sensitivity for the specific detection of low levels of Pneumocystis jirovecii. To do so, two primer sets were designed, targeting the family of genes that code for the most abundant surface protein of Pneumocystis spp., namely the major surface glycoproteins (Msg), and the mitochondrial large subunit rRNA (mtLSUrRNA) multicopy gene, simultaneously detecting two regions. PCR methods are instrumental in detecting these low levels; however, current nested-PCR methods are time-consuming and complex. To validate our new real-time Msg-A/mtLSUrRNA PCR protocol, we compared it with nested-PCR based on the detection of Pneumocystis mitochondrial large subunit rRNA (mtLSUrRNA), one of the main targets used to detect this pathogen. All samples identified as positive by the nested-PCR method were found positive using our new real-time PCR protocol, which also detected P. jirovecii in three nasal aspirate samples that were negative for both rounds of nested-PCR. Furthermore, we read both rounds of the nested-PCR results for comparison and found that some samples with no PCR amplification, or with a feeble band in the first round, correlated with higher Ct values in our real-time Msg-A/mtLSUrRNA PCR. This finding demonstrates the ability of this new single-round protocol to detect low Pneumocystis levels. This new assay provides a valuable alternative for P. jirovecii detection, as it is both rapid and sensitive.

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Identifying the etiologic role of Parvovirus B19 in non-immune hydrops fetalis by histopathology, immunohistochemistry and nucleic acid testing: a retrospective study

Open Medicine ◽

10.2478/s11536-007-0029-z ◽

2007 ◽

Vol 2 (3) ◽

pp. 271-279 ◽

Cited By ~ 1

Author(s):

Koray Ergunay ◽

Gulcin Altinok ◽

Bora Gurel ◽

Ahmet Pinar ◽

Arzu Sungur ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

San Francisco ◽

Real Time Pcr ◽

Nested Pcr ◽

Parvovirus B19 ◽

Etiologic Agent ◽

Hydrops Fetalis ◽

Nucleic Acid Detection

AbstractIntrauterine Parvovirus B19 infections may cause fetal anemia, non-immune hydrops fetalis or abortion. This study focuses on the pathogenic role of Parvovirus B19 in non-immune hydrops fetalis at Hacettepe University, a major reference hospital in Turkey. Twenty-two cases of non-immune hydrops fetalis were retrospectively selected out of a total of 431 hydrops fetalis specimens from the Department of Pathology archieves. Paraffine embedded tissue sections from placental and liver tissues from each case were evaluated by histopathology, immunohistochemistry, nested PCR and commercial quantitative Real-time PCR. Viral DNA was detected in placental tissues by Real-time PCR in 2 cases (2/22, 9.1%) where histopathology also revealed changes suggestive of Parvovirus B19 infection. No significant histopathologic changes were observed for the remaining sections. Nested PCR that targets the VP1 region of the viral genome and immunohistochemistry for viral capsid antigens were negative for all cases. As a result, Parvovirus B19 is identified as the etiologic agent for the development of non-immune hydrops fetalis for 9.1% of the cases in Hacettepe University, Turkey. Real-time PCR is observed to be an effective diagnostic tool for nucleic acid detection from paraffine embedded tissues. Part of this study was presented as a poster at XIIIth International Congress of Virology, San Francisco, USA (Abstract V-572).

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Comparison of QIAsymphony Automated and QIAamp Manual DNA Extraction Systems for Measuring Epstein-Barr Virus DNA Load in Whole Blood Using Real-Time PCR

Journal of Molecular Diagnostics ◽

10.1016/j.jmoldx.2011.07.006 ◽

2011 ◽

Vol 13 (6) ◽

pp. 695-700 ◽

Cited By ~ 10

Author(s):

Stella Laus ◽

Lawrence A. Kingsley ◽

Michael Green ◽

Robert M. Wadowsky

Keyword(s):

Real Time ◽

Dna Extraction ◽

Real Time Pcr ◽

Whole Blood ◽

Epstein Barr Virus ◽

Barr Virus ◽

Epstein Barr

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Performance Evaluation of the Real-Q Cytomegalovirus (CMV) Quantification Kit Using Two Real-Time PCR Systems for Quantifying CMV DNA in Whole Blood

Annals of Laboratory Medicine ◽

10.3343/alm.2016.36.6.603 ◽

2016 ◽

Vol 36 (6) ◽

pp. 603-606 ◽

Cited By ~ 4

Author(s):

Jong Eun Park ◽

Ji-Youn Kim ◽

Sun Ae Yun ◽

Myoung-Keun Lee ◽

Hee Jae Huh ◽

...

Keyword(s):

Performance Evaluation ◽

Real Time ◽

Real Time Pcr ◽

Whole Blood ◽

The Real

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Detection and Quantification of Human Adenoviruses in Surface Waters by Nested PCR, TaqMan Real-Time PCR and Cell Culture Assays

Water Air & Soil Pollution ◽

10.1007/s11270-007-9608-5 ◽

2007 ◽

Vol 191 (1-4) ◽

pp. 83-93 ◽

Cited By ~ 27

Author(s):

M. Muscillo ◽

M. Pourshaban ◽

M. Iaconelli ◽

S. Fontana ◽

A. Di Grazia ◽

...

Keyword(s):

Cell Culture ◽

Real Time ◽

Surface Waters ◽

Real Time Pcr ◽

Nested Pcr ◽

Human Adenoviruses ◽

Detection And Quantification

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Efficient nested-PCR-based method development for detection and genotype identification of Acanthamoeba from a small volume of aquatic environmental sample

Scientific Reports ◽

10.1038/s41598-021-00968-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tsui-Kang Hsu ◽

Jung-Sheng Chen ◽

Hsin-Chi Tsai ◽

Chi-Wei Tao ◽

Yu-Yin Yang ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Nested Pcr ◽

In Silico ◽

Environmental Samples ◽

Method Development ◽

Small Volume ◽

Detection Efficiency ◽

Genotyping Method ◽

Pcr Method

AbstractAcanthamoeba spp. are opportunistic human pathogens that cause granulomatous amoebic encephalitis and keratitis, and their accurate detection and enumeration in environmental samples is a challenge. In addition, information regarding the genotyping of Acanthamoeba spp. using various PCR methods is equally critical. Therefore, considering the diverse niches of habitats, it is necessary to develop an even more efficient genotyping method for Acanthamoeba spp. detection. This study improved the sensitivity of detection to avoid underestimation of Acanthamoeba spp. occurrence in aquatic environmental samples, and to accurately define the pathogenic risk by developing an efficient PCR method. In this study, a new nested genotyping method was established and compared with various PCR-based methods using in silico, lab, and empirical tests. The in silico test showed that many PCR-based methods could not successfully align specific genotypes of Acanthamoeba, except for the newly designed nested PCR and real-time PCR method. Furthermore, 52 water samples from rivers, reservoirs, and a river basin in Taiwan were analysed by six different PCR methods and compared for genotyping and detection efficiency of Acanthamoeba. The newly developed nested-PCR-based method of genotyping was found to be significantly sensitive as it could effectively detect the occurrence of Acanthamoeba spp., which was underestimated by the JDP-PCR method. Additionally, the present results are consistent with previous studies indicating that the high prevalence of Acanthamoeba in the aquatic environment of Taiwan is attributed to the commonly found T4 genotype. Ultimately, we report the development of a small volume procedure, which is a combination of recent genotyping PCR and conventional real-time PCR for enumeration of aquatic Acanthamoeba and acquirement of biologically meaningful genotyping information. We anticipate that the newly developed detection method will contribute to the precise estimation, evaluation, and reduction of the contamination risk of pathogenic Acanthamoeba spp., which is regularly found in the water resources utilised for domestic purposes.

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Quantitative detection ofHelicobacter pyloriin water samples by real-time PCR amplification of the cag pathogenicity island gene,cagE

Journal of Applied Microbiology ◽

10.1111/j.1365-2672.2009.04219.x ◽

2009 ◽

Vol 107 (2) ◽

pp. 416-424 ◽

Cited By ~ 10

Author(s):

M.A. Yáñez ◽

V.M. Barberá ◽

E. Soria ◽

V. Catalán

Keyword(s):

Real Time ◽

Water Samples ◽

Real Time Pcr ◽

Pcr Amplification ◽

Pathogenicity Island ◽

Quantitative Detection ◽

Cag Pathogenicity Island

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Real-Time PCR Quantification of Vibrio parahaemolyticus in Oysters Using an Alternative Matrix

Journal of Food Protection ◽

10.4315/0362-028x-67.11.2424 ◽

2004 ◽

Vol 67 (11) ◽

pp. 2424-2429 ◽

Cited By ~ 38

Author(s):

G. E. KAUFMAN ◽

G. M. BLACKSTONE ◽

M. C. L. VICKERY ◽

A. K. BEJ ◽

J. BOWERS ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Vibrio Parahaemolyticus ◽

Pcr Amplification ◽

Oyster Tissue ◽

Pcr Assay ◽

Colony Hybridization ◽

The Real ◽

Mantle Fluid ◽

June July

This study examined the relationship between levels of total Vibrio parahaemolyticus found in oyster tissues and mantle fluid with the goal of using mantle fluid as a template matrix in a new quantitative real-time PCR assay targeting the thermolabile hemolysin (tlh) gene for the enumeration of total V. parahaemolyticus in oysters. Oysters were collected near Mobile Bay, Ala., in June, July, and September and tested immediately after collection and storage at 26°C for 24 h. Initial experiments using DNA colony hybridization targeting tlh demonstrated that natural V. parahaemolyticus levels in the mantle fluid of individual oysters were strongly correlated (r = 0.85, P < 0.05) with the levels found in their tissues. When known quantities of cultured V. parahaemolyticus cells were added to real-time PCR reactions that contained mantle fluid and oyster tissue matrices separately pooled from multiple oysters, a strong linear correlation was observed between the real-time PCR cycle threshold and the log concentration of cells inoculated into each PCR reaction (mantle fluid: r = 0.98, P < 0.05; and oyster: r = 0.99, P < 0.05). However, the mantle fluid exhibited less inhibition of the PCR amplification than the homogenized oyster tissue. Analysis of natural V. parahaemolyticus populations in mantle fluids using both colony hybridization and real-time PCR demonstrated a significant (P < 0.05) but reduced correlation (r =−0.48) between the two methods. Reductions in the efficiency of the real-time PCR that resulted from low population densities of V. parahaemolyticus and PCR inhibitors present in the mantle fluid of some oysters (with significant oyster-to-oyster variation) contributed to the reduction in correlation between the methods that was observed when testing natural V. parahaemolyticus populations. The V. parahaemolyticus–specific real-time PCR assay used for this study could estimate elevated V. parahaemolyticus levels in oyster mantle fluid within 1 h from sampling time.

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Detection and Enumeration of Aromatic Oxygenase Genes by Multiplex and Real-Time PCR

Applied and Environmental Microbiology ◽

10.1128/aem.69.6.3350-3358.2003 ◽

2003 ◽

Vol 69 (6) ◽

pp. 3350-3358 ◽

Cited By ~ 198

Author(s):

Brett R. Baldwin ◽

Cindy H. Nakatsu ◽

Loring Nies

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Gene Copy Number ◽

Pcr Amplification ◽

Gene Copy ◽

Hydrocarbon Biodegradation ◽

Polymerization Temperature ◽

Primer Sets ◽

The Impact

ABSTRACT Our abilities to detect and enumerate pollutant-biodegrading microorganisms in the environment are rapidly advancing with the development of molecular genetic techniques. Techniques based on multiplex and real-time PCR amplification of aromatic oxygenase genes were developed to detect and quantify aromatic catabolic pathways, respectively. PCR primer sets were identified for the large subunits of aromatic oxygenases from alignments of known gene sequences and tested with genetically well-characterized strains. In all, primer sets which allowed amplification of naphthalene dioxygenase, biphenyl dioxygenase, toluene dioxygenase, xylene monooxygenase, phenol monooxygenase, and ring-hydroxylating toluene monooxygenase genes were identified. For each primer set, the length of the observed amplification product matched the length predicted from published sequences, and specificity was confirmed by hybridization. Primer sets were grouped according to the annealing temperature for multiplex PCR permitting simultaneous detection of various genotypes responsible for aromatic hydrocarbon biodegradation. Real-time PCR using SYBR green I was employed with the individual primer sets to determine the gene copy number. Optimum polymerization temperatures for real-time PCR were determined on the basis of the observed melting temperatures of the desired products. When a polymerization temperature of 4 to 5Ã¯Â¿Â½C below the melting temperature was used, background fluorescence signals were greatly reduced, allowing detection limits of 2 Ã¯Â¿Â½ 102 copies per reaction mixture. Improved in situ microbial characterization will provide more accurate assessment of pollutant biodegradation, enhance studies of the ecology of contaminated sites, and facilitate assessment of the impact of remediation technologies on indigenous microbial populations.

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