Development and evaluation of a real-time polymerase chain reaction for fast diagnosis of sporotrichosis caused by Sporothrix globosa

2019 ◽  
Vol 58 (1) ◽  
pp. 61-65 ◽  
Author(s):  
Mingrui Zhang ◽  
Fuqiu Li ◽  
Jie Gong ◽  
Xin Yang ◽  
Jianzhong Zhang ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Specific Method ◽  
Chain Reaction ◽  
Clinical Specimens ◽  
Sporothrix Globosa ◽  
The Real ◽  
Pcr Method ◽  
Polymerase Chain

Abstract Sporothrix globosa is an important clinical pathogen in the Sporothrix complex, which is causing sporotrichosis. S. globosa is distributed worldwide, especially in Asia. The transmission medium of S. globosa is mainly contaminated soil or decaying vegetation, and the infection usually caused by transcutaneous trauma, through which the fungal conidia or yeast cells enter the host. Although the clinical manifestations of sporotrichosis caused by S. globosa is always benign, there have been several outbreaks worldwide. In this study, we established a novel real-time polymerase chain reaction (PCR) method based on the internal transcribed spacer (ITS) sequence for the identification of S. globosa. The assay was further evaluated by clinical specimens obtained from patients of sporotrichosis. The sensitivity and specificity of the real-time PCR method was both 100%. The detection limit was 10 fg. The positive detection rate for 30 clinical specimens, which were confirmed infected by S. globosa, was 100%. The real-time PCR method established in this paper is a rapid, sensitive and specific method for the identification of S. globosa. It can detect S. globosa in clinical specimen from patients with sporotrichosis, which is helpful for fast clinical diagnosis.

Quantitative Real-Time Polymerase Chain Reaction Detection of BK Virus Using Labeled Primers

2010 ◽  
Vol 134 (3) ◽  
pp. 444-448 ◽  
Author(s):  
Zhengming Gu ◽  
Jianmin Pan ◽  
Matthew J. Bankowski ◽  
Randall T. Hayden
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Bk Virus ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

Quantitative assessment of hematopoietic chimerism after bone marrow transplantation by real-time quantitative polymerase chain reaction

Blood ◽  
2002 ◽  
Vol 99 (12) ◽  
pp. 4618-4625 ◽  
Author(s):  
Mehdi Alizadeh ◽  
Marc Bernard ◽  
Bruno Danic ◽  
Charly Dauriac ◽  
Brigitte Birebent ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Stem Cell ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Assessment ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Hematopoietic Stem ◽  
Pcr Method ◽  
Polymerase Chain

We have developed a real-time quantitative polymerase chain reaction (PCR) assay using TaqMan technology (Applied Biosystems, Foster City, CA) for monitoring donor cell engraftment in allogenic hematopoietic stem cell transplant recipients. For this purpose, we selected 19 specific sequence polymorphisms belonging to 11 human biallelic loci located on 9 different chromosomes. Using a set of specially designed primers and fluorogenic probes, we evaluated the 19 markers' informativity on a panel of 126 DNA samples from 63 recipient/donor pairs. In more than 90% of these pairs, discrimination between recipient and donor genetic profile was possible. By using serial dilutions of mixed DNAs, we evaluated the linearity and sensitivity of the method. A linear correlation with rhigher than 0.98 and a sensitivity of 0.1% proved reproducible. Fluorescent-based PCR of short tandem repeats (STR-PCR) and real-time PCR chimerism assay were compared with a panel of artificial cell mixtures. The main advantage of the real-time PCR method over STR-PCR chimerism assays is the absence of PCR competition and plateau biases, and results evidenced greater sensitivity and linearity with the real-time PCR method. Furthermore, different samples can be tested in the same PCR run with a final result in fewer than 48 hours. Finally, we prospectively analyzed patients who received allografts and present 4 different clinical situations that illustrate the informativity level of our method. In conclusion, this new assay provides an accurate quantitative assessment of mixed chimerism that can be useful in guiding early implementation of additional treatments in hematopoietic stem cell transplantation.

scholarly journals Deteksi Plasmodium falciparum dengan menggunakan metode real-time polymerase chain reaction di daerah Likupang dan Bitung

2016 ◽  
Vol 4 (1) ◽  
Author(s):  
David C. Tooy ◽  
Janno B. Bernadus ◽  
Angle Sorisi
Keyword(s):  
Polymerase Chain Reaction ◽  
Plasmodium Falciparum ◽  
Real Time ◽  
Real Time Pcr ◽  
18S Rrna ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Negative Results ◽  
Pcr Method ◽  
Polymerase Chain

Abstract: Malaria is one of the most important parasitic disease which is caused by Plasmodium spp. There are approximately 1,2 billion people in the world with high risk of getting malaria. Plasmodium falciparum (P. falciparum) is the cause of tropical malaria or falciparum malaria, and is responsible for most of the mortality rate. Currently, real-time polymerase chain reaction (RT-PCR) is being studied as an alterative of conventional malarian examination. Mangold et al reported that RT-PCR have 94.1% sensitivity and 100% specificity compared to microscopic examination in detecting P. falciparum. The aim of this research is to detect the presence of P. falciparum using RT-PCR in Likupang and Bitung region. This research were using descriptive design to find out the capability of real-time PCR method to detect P. falciparum in Likupang dan Bitung region. The researcher have examined 71 samples which are fulfill the research sample’s criteria. Postive results of P. falciparum found in 18 samples (25,3%) and negative results in 53 samples (74,6%) of total 71 samples with using RT-PCR. No positive results were found in samples from Likupang. There are positive result of P. falciparum in samples from Bitung. It is concluded that RT-PCR method can detect the presence of P. falciparum from the samples obtained from Likupang and Bitung based on the presence of its DNA. This detection efford is done by using 18S rRNA as target gene and ajust specific temperature on the RT-PCR instrument.Keywords: Plasmodium falciparum, Real-time Polymerase Chain Reaction (PCR), DetectionAbstrak: Malaria merupakan salah satu penyakit penting yang disebabkan oleh parasit Plasmodium spp. Kira-kira 1,2 miliar penduduk dunia memiliki risiko tinggi untuk mendapat malaria. Di Indonesia sendiri, terdapat 343.527 kasus terkonfirmasi dan 45 kematian karena malaria. Plasmodium falciparum (P. Falciparum) merupakan penyebab dari malaria tropika atau malaria falsiparum, dan bertanggung jawab atas sebagian besar angka mortalitas. Saat ini Real-Time Polymerase Chain Reaction (RT-PCR) telah banyak diteliti sebagai alternatif dari pemeriksaan malaria. Mangold dkk melaporkan bahwa real-time PCR memiliki nilai sensitivitas 94,1% dan nilai spesifisitas 100% terhadap pemeriksaan mikroskopis dalam mendeteksi P. falciparum. Penelitian bertujuan untuk mendeteksi P. falciparum dengan menggunakan RT-PCR di daerah Likupang dan Bitung. Penelitian ini menggunakan rancangan penelitian deskriptif untuk mengetahui kemampuan metode real-time PCR dalam mendeteksi P. falciparum di daerah Likupang dan Bitung. Tujuan penelitian ini ialah untuk mendeteksi keberadaan P. falciparum dengan menggunakan metode real-time PCR di daerah Likupang dan Bitung. Peneliti memeriksa 71 sampel darah yang memenuhi kriteria sampel penelitian. Hasil positif P. falciparum ditemukan pada 18 sampel (25,3 %) dan hasil negatif pada 53 sampel (74,6 %) dari total 71 sampel dengan menggunakan RT-PCR. Tidak ditemukannya hasil positif P. falciparum pada sampel dari Likupang. Ditemukan hasil positif P. falciparum pada sampel dari Bitung. Simpulan: Metode RT-PCR dapat mendeteksi P. falciparum berdasarkan keberadaan DNA-nya pada sampel yang diperoleh dari daerah Likupang dan Bitung. Deteksi ini berhasil dilakukan dengan menggunakan 18S rRNA sebagai gen target dan pengaturan suhu tertentu pada instrument RT-PCR.Kata kunci: P. falciparum, Real-time Polymerase Chain Reaction (PCR), Detection

scholarly journals Molecular diagnosis of Anaplasma marginale in cattle: quantitative evaluation of a real-time PCR (Polymerase Chain Reaction) based on msp5 gene

2014 ◽  
Vol 34 (1) ◽  
pp. 29-33 ◽  
Author(s):  
Gisele M. Bacanelli ◽  
Carlos A. N. Ramos ◽  
Flábio R. Araújo
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Anaplasma Marginale ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Chain Reaction ◽  
The Real ◽  
End Point ◽  
Polymerase Chain

The rickettsia Anaplasma marginale is considered the main agent of bovine anaplasmosis. Due the nonspecific clinical signs of the anaplasmosis, the diagnosis of infection depends of laboratory confirmation. In recent years, molecular diagnostic methods have been used to detect A. marginale in cattle. However, the existence of a large number of assays of different sensitivity and cost makes the choice of an appropriate test difficult. In the present study, a real-time Polymerase Chain Reaction (PCR) based on the msp5 target gene was quantitatively assessed and compared to an end point PCR. Both reactions were subjected to sensitivity and specificity evaluation using plasmid DNA and samples from cattle experimentally infected with A. marginale. A comparative field trial of the tests was carried out using samples of cattle from a stable enzootic area for A. marginale. The real-time PCR showed a higher sensitivity than the end point PCR. This reaction (i.e. real-time PCR) was able to detect one copy of the msp5 gene in 100 ηg of plasmidial DNA, and more than 80% of its results were positive among experimentally infected animals seven days after infection. In addition, based on in silico analysis, the real-time PCR evaluated in the present study appears to be useful for the detection of A. ovis.

scholarly journals Quantification of Magnaporthe grisea During Infection of Rice Plants Using Real-Time Polymerase Chain Reaction and Northern Blot/Phosphoimaging Analyses

2002 ◽  
Vol 92 (8) ◽  
pp. 870-876 ◽  
Author(s):  
Min Qi ◽  
Yinong Yang
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Northern Blot ◽  
Host Resistance ◽  
Real Time Pcr ◽  
Magnaporthe Grisea ◽  
Chain Reaction ◽  
Fungal Pathogenicity ◽  
The Real ◽  
Polymerase Chain

Rice blast, caused by Magnaporthe grisea, is a serious fungal disease of rice worldwide. Currently, evaluation of the fungal pathogenicity and host resistance is mainly based on a disease rating or measurement of blast lesion number and size. However, these methods only provide visual estimation rather than accurate measurement of fungal growth in rice plants. In this study, DNA-based real-time polymerase chain reaction (PCR) and RNA-based northern blot/phosphoimaging analyses were evaluated to quantify M. grisea. Both methods were sensitive, specific, and reproducible and could accurately measure the relative growth and absolute biomass of M. grisea. The real-time PCR analysis showed that the growth of M. grisea in seedling leaves of susceptible cultivars (M201 and Wells) was ≈46 to 80 times higher than that of a resistant cultivar (Drew) at 4 and 6 days after inoculation. The data obtained from the real-time PCR assays also were consistent with that from northern blot/ phosphoimaging analysis. However, the real-time PCR approach was much faster and more convenient in most cases. Therefore, it is an excellent tool for in planta quantification of M. grisea and can be used for reliable assessment of fungal pathogenicity and host resistance

scholarly journals Detection of Schistosoma mansoni DNA using polymerase chain reaction from serum and dried blood spot card samples of an adult population in North-western Tanzania

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Antje Fuss ◽  
Humphrey D. Mazigo ◽  
Andreas Mueller
Keyword(s):  
Polymerase Chain Reaction ◽  
Schistosoma Mansoni ◽  
Real Time ◽  
Real Time Pcr ◽  
Rank Test ◽  
Specific Method ◽  
Chain Reaction ◽  
Serum Samples ◽  
Severe Infections ◽  
Polymerase Chain

Abstract Background Real-time polymerase chain reaction (PCR) is a sensitive and specific method for diagnosing schistosomiasis. However, this method should be performed in a laboratory, usually located distant from the sample collection site. Therefore, it is important to have fast sampling preservation methods, which allow simple transport prior to DNA extraction and amplification. The aim of this study was to verify if blood samples applied to filter paper are suitable for analysis of Schistosoma mansoni DNA by real-time PCR. Methods A cross-sectional study was conducted among 100 study participants aged 17 to 70 years in a fishing village on the southern shore of Lake Victoria, Tanzania. Serum samples and ethylenediaminetetraacetic acid (EDTA)-anticoagulated whole blood for preparation of dried blood spots (DBS) were collected to test for Schistosoma mansoni infection by real-time PCR. A combined diagnostic reference of positive results of serum-based real-time PCR and the Kato-Katz (KK) method was used for analysis. Sensitivity and negative predictive value (NPV) were calculated. The Wilcoxon signed-rank test was chosen to compare the mean cycle threshold (Ct) values from serum and DBS. Results According to the reference, 92.5% S. mansoni positive samples were determined. The serum-based real-time PCR performed excellently with 95.4% sensitivity, whereas the DBS-based real-time PCR showed a low sensitivity (45.4%). The Ct-values were significantly higher in DBS (median: 37.3) than in serum samples (median: 27.5, P < 0.001), reflecting a lower parasite-specific DNA load on the filter cards. With increasing egg counts, an increase in sensitivity was observed for all methods. The POC-CCA test and the serum-based real-time PCR showed a sensitivity of 100% for medium and severe infections. The DBS real-time PCR showed a sensitivity of only 85.7% even for severe infections. Conclusions DBS-based real-time PCR did not provide good results in our study and therefore should not be recommended or must be tested concerning temperature of storage, storage duration, use of different filter papers and extraction methods before it is used in future studies. In contrast, our results showed that the POC-CCA test is a sensitive and precise test for detecting S. mansoni infections "Image missing" .

scholarly journals Validation of a Real-Time Polymerase Chain Reaction Assay for the Identification of Meloidogyne arenaria

Plant Disease ◽  
2011 ◽  
Vol 95 (7) ◽  
pp. 835-838 ◽  
Author(s):  
Paula Agudelo ◽  
Stephen A. Lewis ◽  
Bruce A. Fortnum
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Soil Samples ◽  
Meloidogyne Arenaria ◽  
Chain Reaction ◽  
Pcr Assay ◽  
The Real ◽  
Polymerase Chain ◽  
Species Specific

Meloidogyne arenaria is an economically important parasite of many crops worldwide. Identification and detection of this species in soil samples is necessary for the design of crop rotation systems, selection of resistant cultivars, and potential use of biological control options. The objective of this study was to develop and validate a real-time polymerase chain reaction (PCR) assay, using species-specific primers and SYBR Green I Dye, for identification of M. arenaria. The specificity of the assay was confirmed by testing for amplification of DNA from other Meloidogyne spp. and from M. arenaria populations of different geographic origins. Field soil samples containing a mixture of M. arenaria and M. incognita were used to compare identification by the real-time PCR assay with identification by esterase phenotype analysis of mature females and by morphometrics of juveniles. The real-time PCR assay provided an accurate and sensitive means for the identification of single juveniles from soil samples.

scholarly journals Sensitive Detection of Fusarium circinatum in Pine Seed by Combining an Enrichment Procedure with a Real-Time Polymerase Chain Reaction Using Dual-Labeled Probe Chemistry

2009 ◽  
Vol 99 (5) ◽  
pp. 582-590 ◽  
Author(s):  
Renaud Ioos ◽  
Céline Fourrier ◽  
Gabriela Iancu ◽  
Thomas R. Gordon
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Fusarium Circinatum ◽  
Conventional Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
The Real ◽  
Polymerase Chain ◽  
Seed Dna

Fusarium circinatum is the causal agent of pitch canker disease on numerous Pinus spp. This aggressive fungus may infect pine seed cryptically and, therefore, can easily be spread long distances by the seed trade. F. circinatum has recently been listed as a quarantine organism in numerous countries throughout the world, which prompted the development of a specific and sensitive tool for the detection of this pathogen in conifer seed. A new detection protocol for F. circinatum based on a biological enrichment step followed by a real-time polymerase chain reaction (PCR) assay was developed. Several enrichment protocols were compared and a 72-h incubation of the seed with potato dextrose broth was the most efficient technique to increase F. circinatum biomass before DNA extraction. The relative accuracy, specificity, and sensitivity of the real-time PCR assay was evaluated in comparison with a previously published conventional PCR test on 420 seed DNA extracts. The real-time PCR described here proved to be highly specific and significantly more sensitive than the conventional PCR, and enabled the detection of F. circinatum in samples artificially contaminated with less than 1/1,000 infected seed, as well as in naturally infected samples. Last, in order to routinely check the quality of the seed DNA extracts, a primer–probe combination that targets a highly conserved region within the 18S ribosomal DNA in plants or fungi was successfully developed. This assay allows for quick and reliable detection of F. circinatum in seed, which can help to prevent long-distance spread of the pathogen via contaminated seed lots.

scholarly journals Development of Real-Time Polymerase Chain Reaction Assay with TaqMan Probe for the Quantitative Detection of Infectious Hypodermal and Hematopoietic Necrosis Virus from Shrimp

2006 ◽  
Vol 89 (1) ◽  
pp. 240-244 ◽  
Author(s):  
Zhi-Qin Yue ◽  
Hong Liu ◽  
Wei-Ji Wang ◽  
Zhi-Wen Lei ◽  
Cheng-Zhu Liang ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Taqman Probe ◽  
Quantitative Detection ◽  
Necrosis Virus ◽  
Chain Reaction ◽  
Pcr Assay ◽  
The Real ◽  
Polymerase Chain

Abstract An assay was developed for the detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) based on real-time quantitative polymerase chain reaction (PCR). A pair of primers and a TaqMan probe were designed that are specific for the recognition of a conservative region in the IHHNV genome. The IHHNV real-time PCR assay had a detection limit of 9 DNA copies,with a dynamic range of detection between 9 106 and 9 DNA copies. The primer pairs and probe were specific to IHHNV and did not cross-reactwith shrimp genomic DNAor other shrimp viruses such as White Spot Syndrome Virus (WSSV), Monodon Baculovirus (MBV), and hepatopancreatic parvovirus (HPV). This assay has a broad application for basic and clinical investigations. For clinical samples, the real-time PCR assay detected all the positive samples screened by conventional PCR, which indicated the sensitivity of the real-time assay. The IHHNV real-time PCR assay with high sensitivity, specificity, wide range of detection ability, and simplicity is particularly useful for screening large numbers of specimens and measuring viral loads to monitor the broodstock.

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