scholarly journals Differential Diagnosis ofEntamoebaspp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Thiago dos Santos Gomes ◽  
Mariana Coimbra Garcia ◽  
Flavia de Souza Cunha ◽  
Heloisa Werneck de Macedo ◽  
José Mauro Peralta ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Intestinal Parasites ◽  
Laboratory Diagnosis ◽  
Sybr Green ◽  
Similar Species ◽  
Green Is ◽  
Stool Samples ◽  
Polymerase Chain ◽  
Negative Controls

Amoebiasis, a disease caused byEntamoeba histolytica, is usually diagnosed by microscopic examination, which does not differentiate the morphologically identical species of theE. histolytica/E. disparcomplex. Furthermore, morphologically similar species such asEntamoeba hartmannicontribute to misidentification. Therefore, there is a need for more sensitive and specific methods. This study standardized a multiplex real-time PCR system forE. histolyticaandE. disparand a single real-time PCR forE. hartmanni. The multiplex protocol detected up to 0.0143 pg ofE. histolyticaDNA and 0.5156 pg ofE. disparDNA, and the average melting temperature (Tm) was 73°C and 70°C, respectively. ForE. hartmanni, theTmwas 73°C and the amplification was successful down to 0.03 fg of plasmid DNA. Negative controls and other intestinal parasites presented no amplification. Among the 48 samples tested,E. disparDNA was detected in 37; none exhibitedE. histolyticaDNA and 11 were negative in the multiplex protocol. In 4 of these 11 samples, however,E. hartmanniDNA was amplified. SYBR Green is demonstrated to be an interesting option and these combined PCR reactions can improve laboratory diagnosis of amoebiasis in developing countries.

scholarly journals Molecular Characterization of Giardia duodenalis in Children and Adults Sampled in Algeria

2020 ◽  
Vol 9 (1) ◽  
pp. 54
Author(s):  
Salem Belkessa ◽  
Daniel Thomas-Lopez ◽  
Karim Houali ◽  
Farida Ghalmi ◽  
Christen Rune Stensvold
Keyword(s):  
Real Time ◽  
Molecular Characterization ◽  
Real Time Pcr ◽  
Intestinal Parasites ◽  
Giardia Duodenalis ◽  
Specific Pcr ◽  
Stool Samples ◽  
Pcr Targeting ◽  
Assemblage B

The molecular epidemiology of giardiasis in Africa remains unclear. A study was carried out across four hospitals in Algeria. A total of 119 fecal samples from 55 children, 37 adults, and 27 individuals of undetermined age, all scored positive for intestinal parasites by microscopy, and were screened by real-time PCR for Giardia. Molecular characterization of Giardia was performed by assemblage-specific PCR and PCR targeting the triose phosphate isomerase gene (tpi). Of the 119 samples, 80 (67%) were Giardia-positive by real-time PCR. For 48 moderately-highly real-time PCR-positive samples, tpi genotyping assigned 22 samples to Assemblage A and 26 to Assemblage B. Contrary to Assemblage A, Assemblage B exhibited substantial genetic diversity and allelic heterozygosity. Assemblage-specific PCR proved to be specific for discriminating Assemblage A or B but not as sensitive as tpi genotyping. We confirmed that real-time PCR is more sensitive than microscopy for detecting Giardia in stool samples and that robust amplification and sequencing of the tpi gene is feasible when moderate-to-strongly real-time PCR-positive samples are used. This study is one of the few performed in Africa providing genotyping data on Giardia infections in humans. Both assemblages A and B were commonly seen and not associated with specific sociodemographic data.

scholarly journals Development of Conventional Multiplex PCR: A Rapid Technique for Simultaneous Detection of Soil-Transmitted Helminths

Pathogens ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 152 ◽  
Author(s):  
Vivornpun Sanprasert ◽  
Ruthairat Kerdkaew ◽  
Siriporn Srirungruang ◽  
Sarit Charuchaibovorn ◽  
Kobpat Phadungsaksawasdi ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Real Time ◽  
Real Time Pcr ◽  
Intestinal Parasites ◽  
Simultaneous Detection ◽  
Diagnostic Tools ◽  
Multiple Infections ◽  
Parasitic Infections ◽  
Soil Transmitted Helminths ◽  
Stool Samples

Soil-transmitted helminths (STHs) are the most common intestinal parasites infecting humans worldwide. STH infections are a major cause of morbidity and disability. Accurate diagnostic tools are pivotal for assessing the exact prevalence of parasitic infections. Microscopic examination and culture techniques have been used to observe the presence of eggs or larvae of parasites in stool samples, but they are time-consuming and have low sensitivity. Therefore, accurate, simple, and inexpensive diagnostic techniques are still required for simultaneous detection of STH infections. Although molecular-based techniques, such as real-time PCR and multiplex real-time PCR, have been developed, they are not suitable for routine diagnosis due to the requirement for expensive reagents and instruments. In this study, we established a conventional multiplex PCR for simultaneous rapid detection of Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis in stool samples. Our results show that the multiplex PCR could detect the DNA of STHs at a very low target gene concentrations (lower than 1 pg) with no cross-amplification. Multiplex PCR had five times higher sensitivity than the formalin–ethyl acetate concentration technique (FECT) in the detection of multiple infections, and two times higher for detection of S. stercoralis. However, multiplex PCR was comparable to FECT in the detection of A. lumbricoides and N. americanus. In conclusion, this method could be used as an alternative method for the detection of STHs, especially for S. stercoralis.

scholarly journals Detection and species identification of microsporidial infections using SYBR Green real-time PCR

2011 ◽  
Vol 60 (4) ◽  
pp. 459-466 ◽  
Author(s):  
Spencer D. Polley ◽  
Samuel Boadi ◽  
Julie Watson ◽  
Alan Curry ◽  
Peter L. Chiodini
Keyword(s):  
Real Time ◽  
Species Identification ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Enterocytozoon Bieneusi ◽  
Sybr Green ◽  
Pcr Assay ◽  
Real Time Analysis ◽  
Highly Sensitive ◽  
Stool Samples

Diagnosis of microsporidial infections is routinely performed by light microscopy, with unequivocal non-molecular species identification achievable only through electron microscopy. This study describes a single SYBR Green real-time PCR assay for the simultaneous detection and species identification of such infections. This assay was highly sensitive, routinely detecting infections containing 400 parasites (g stool sample)−1, whilst species identification was achieved by differential melt curves on a Corbett Life Science Rotor-Gene 3000. A modification of the QIAamp DNA tissue extraction protocol allowed the semi-automated extraction of DNA from stools for the routine diagnosis of microsporidial infection by real-time PCR. Of 168 stool samples routinely analysed for microsporidian spores, only five were positive by microscopy. By comparison, 17 were positive for microsporidial DNA by real-time analysis, comprising 14 Enterocytozoon bieneusi, one Encephalitozoon cuniculi and two separate Pleistophora species infections.

scholarly journals Perbandingan Metode SYBR Green dan Hydrolysis Probe dalam Analisis DNA Gelatin Sapi dan Gelatin Babi Menggunakan Real Time Polymerase Chain Reaction

2017 ◽  
Vol 4 (1) ◽  
pp. 16
Author(s):  
Zilhadia Zilhadia ◽  
Afifah Nurul Izzah ◽  
Ofa Suzanti Betha
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Sybr Green ◽  
Chain Reaction ◽  
Hydrolysis Probe ◽  
Polymerase Chain

Pemanfaatan gelatin secara luas menimbulkan kontroversi dan kekhawatiran bagi masyarakat muslim karena pada umumnya gelatin terbuat dari kulit babi dan sapi. Salah satu teknik analisis yang dapat membedakan gelatin sapi dan gelatin babi adalah Real Time Polymerase Chain Reaction (PCR). Real Time PCR merupakan metode analisis berbasis DNA yang handal, efektif, dan terpecaya. Dalam analisis kualitatif dan kuantitatif, Real Time PCR membutuhkan pewarna fluoresens. Pewarna fluoresens yang umum digunakan adalah SYBR green dan hydrolysis probe. Telah dilakukan perbandingan antara metode SYBR green dan hydrolysis probe dalam analisis DNA gelatin menggunakan Real Time PCR. DNA pada gelatin diisolasi menggunakan kit komersial. Isolat DNA gelatin sapi dan DNA gelatin babi didapatkan sebanyak 19,38 ng/μl dan 13,63 ng/μl dengan kemurnian 1,566 dan 1,573. Isolat DNA yang dianalisis dengan metode SYBR green menggunakan suhu annealing 65o C untuk primer sapi dan suhu annealing 60o C untuk primer babi. Isolat DNA yang dianalisis dengan metode hydrolysis probe menggunakan suhu annealing 60o C untuk primer babi dan primer sapi. Hasil analisis dari kedua metode menunjukkan bahwa metode hydrolysis probe lebih spesifik dalam mengidentifikasi DNA pada gelatin dibandingkan menggunakan metode SYBR green.

scholarly journals Detection of SARS-CoV-2 using real-time polymerase chain reaction in different clinical specimens: A critical review

2021 ◽  
Vol 49 (1) ◽  
pp. 159-164
Author(s):  
Anees Muhammad ◽  
Hajira Ameer ◽  
Syed Adnan Haider ◽  
Ihsan Ali
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Laboratory Diagnosis ◽  
Extraction Methods ◽  
Chain Reaction ◽  
High Detection Rate ◽  
Computed Tomography Scanning ◽  
Rapid Spread ◽  
Polymerase Chain

Coronavirus disease 2019 (COVID-19) is a disease caused by a new strain of coronavirus named as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Globally, since the outbreak, more than seven million confirmed cases of COVID-19 have been reported. The rapid spread and increase in the number of new cases is due to person-to-person transmission. To further control its transmission, early laboratory diagnosis of both asymptomatic and symptomatic patients is crucial. Presently, the COVID-19 diagnosis of infected individuals is dependent on computed tomography scanning and real-time polymerase chain reaction (PCR). The latter is considered more sensitive and efficient for early diagnosis. In this review, general comparisons are made (cases, fatality rate, incubation period, clinical features, and reservoirs) and diag-nostic laboratory procedures (specimens, extraction methods, and positive rates by real-time PCR) are compared between SARS, Middle East Respiratory Syndrome, and SARS-2. In total, 8982 SARS-2 suspected patients specimen data were retrieved, in which 40.9% (n = 3678) were detected as positive by real-time PCR. The specimen-wise high detection rate was observed from bronchoalveolar lavage, followed by saliva, nasal swabs, and sputum. As the COVID-19 cases are persistently increasing, the selection of appropriate specimens and laboratory assay would help in rapid and timely diagnosis.

Real-time PCR using SYBR Green for the detection of Shigella spp. in food and stool samples

2013 ◽  
Vol 27 (1) ◽  
pp. 53-59 ◽  
Author(s):  
W. Mokhtari ◽  
S. Nsaibia ◽  
A. Gharbi ◽  
M. Aouni
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Sybr Green ◽  
Shigella Spp ◽  
Stool Samples

scholarly journals Comparison between Conventional and Real-Time PCR Assays for Diagnosis of Visceral Leishmaniasis

2014 ◽  
Vol 2014 ◽  
pp. 1-4 ◽  
Author(s):  
Mariana R. Pereira ◽  
Fabiana Rocha-Silva ◽  
Cidiane Graciele-Melo ◽  
Camila R. Lafuente ◽  
Telcia Magalhães ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Findings ◽  
Sybr Green ◽  
Polymerase Chain ◽  
Belo Horizonte ◽  
Green System ◽  
Pcr Techniques ◽  
Positive Results

The diagnosis of visceral leishmaniasis (VL) is a challenging issue and several studies worldwide have evaluated the different tools to reach a diagnostic solution. The polymerase chain reaction (PCR) has proven to be effective in detecting the genome ofLeishmaniaspecies in different biological samples. In this study, we compared the conventional PCR and real-time PCR using the Sybr Green system and their application in molecular diagnosis of visceral leishmaniasis in peripheral blood as a biological sample. The genus-specific conserved region of kinetoplast DNA (kDNA) was the target of amplification. We studied 30 samples from patients with suspect of visceral leishmaniasis who were treated by the Medical Clinic of Santa Casa de Belo Horizonte Hospital, Brazil. Among the samples studied, 19 had a confirmed diagnosis for VL by serology and/or by clinical findings. Among these 19 samples, 63% (n=12) presented positive results for serology and 79% (n=15) positive results in both PCR methodologies. This fact suggests that the PCR technique can assist in the diagnosis of visceral leishmaniasis in patients who do not have detectable antibodies by serology but can present the genome of the parasite circulating in whole blood. Also, it was possible to observe that there was conformity between the results of the techniques of cPCR and qPCR using the Sybr Green system in 100% of samples analyzed. These data suggest that both PCR techniques were equally effective for detection of the genome of the parasite in the patient’s blood.

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.

scholarly journals Validation of swab sampling and SYBR Green-based real-time PCR for the diagnosis of Cutaneous Leishmaniasis in French Guiana

2020 ◽  
Author(s):  
Romain Blaizot ◽  
Stéphane Simon ◽  
Marine Ginouves ◽  
Ghislaine Prévot ◽  
Denis Blanchet ◽  
...  
Keyword(s):  
South America ◽  
Cutaneous Leishmaniasis ◽  
Real Time ◽  
Real Time Pcr ◽  
French Guiana ◽  
Laboratory Diagnosis ◽  
Sybr Green ◽  
Non Invasive ◽  
Skin Biopsies ◽  
Sampling Procedures

Recent studies have highlighted the interest of non-invasive sampling procedures coupled to real-time PCR methods for detection of Leishmania species in South America. In French Guiana, sampling method still relied on skin biopsies. Non-invasive protocols should be tested on a large annual cohort to improve routine laboratory diagnosis of Cutaneous Leishmaniasis. Therefore, we evaluated the performances of a new Leishmania detection and species identification protocol involving cotton swabs and SYBR Green real-time PCR of Hsp70 gene, coupled with Sanger sequencing. Between May 2017 and May 2018, 145 patients with ulcerated lesions compatible with Cutaneous Leishmaniasis were included in the Cayenne Hospital and its remote health centres. Each patient underwent scrapings for smear, skin biopsies for parasite culture and PCR-RFLP (RNA pol II) and cotton swabs for SYBR Green PCR. The most accurate diagnostic test was the SYBR Green PCR on swab sampling, showing a 98% sensitivity. Mean PCR cycle threshold (Ct) was of 24.4 Ct (min=17 Ct, max=36 Ct) and was inferior to 35 Ct in 97.6% of samples. All samples positive with real-time PCR SYBR Green were successfully identified at the species level by DNA sequencing. This new method should be considered for routine diagnosis of Cutaneous Leishmaniasis in South America and especially for remote areas, as non-invasive collection tools are easier to use and require less precautions for transportation.

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