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

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.

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Diagnosis and Causative Species of Visceral Leishmaniasis in Southwest Saudi Arabia

American Journal of Tropical Medicine and Hygiene ◽

10.4269/ajtmh.20-1006 ◽

2021 ◽

Author(s):

Aymen Abdelhaleem ◽

Nabil Dhayhi ◽

Mohamed Salih Mahfouz ◽

Ommer Daffalla ◽

Mansour Mubarki ◽

...

Keyword(s):

Saudi Arabia ◽

Visceral Leishmaniasis ◽

Real Time ◽

Real Time Pcr ◽

Leishmania Donovani ◽

Target Genes ◽

Sample Volume ◽

Pcr Products ◽

Polymerase Chain ◽

Robust Evidence

Visceral leishmaniasis (VL) is the most severe clinical form of the disease and has been reported in the Jazan region of southwest Saudi Arabia. This study aimed to diagnose VL by real-time polymerase chain reaction (PCR) and the direct agglutination test (DAT) and to identify the causative Leishmania species. A total of 80 participants, including 30 suspected VL patients, 30 healthy endemic control individuals, and 20 malaria disease controls, were enrolled in this study. Blood samples were collected and tested for Leishmania DNA by real-time PCR and for antibody by the DAT. Sequencing of some amplified PCR products was used to identify the causative Leishmania species. The diagnosis of VL was successfully achieved by both real-time PCR and by DAT with 100% sensitivity. Leishmania donovani and Leishmania infantum species were detected by sequencing both by the kDNA and ITS1 target genes, followed a BLASTn search. The detection of VL antibody by the DAT followed by the confirmatory detection of Leishmania DNA in patient blood by PCR could promote the adoption of the much less invasive and more sensitive methods for the routine diagnosis of VL. Further study with high sample volume to evaluate the PCR and the DAT are needed, to generate more robust evidence. Based on the sequencing results, emerging studies on VL should focus on the causative Leishmania species, reservoirs, and vectors that are important in the study area.

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Field Validation of SYBR Green- and TaqMan-Based Real-Time PCR Using Biopsy and Swab Samples To Diagnose American Tegumentary Leishmaniasis in an Area Where Leishmania (Viannia) braziliensis Is Endemic

Journal of Clinical Microbiology ◽

10.1128/jcm.01954-16 ◽

2016 ◽

Vol 55 (2) ◽

pp. 526-534 ◽

Cited By ~ 22

Author(s):

Ciro Martins Gomes ◽

Mariana Vicente Cesetti ◽

Natália Aparecida de Paula ◽

Sebastián Vernal ◽

Gaurav Gupta ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Sybr Green ◽

Conventional Pcr ◽

Content Type ◽

Tegumentary Leishmaniasis ◽

Precise Diagnosis ◽

American Tegumentary Leishmaniasis ◽

Pcr Techniques ◽

Composite Reference Standard

ABSTRACTThe precise diagnosis of American tegumentary leishmaniasis (ATL) is an essential task due to the disease's associated morbidity. A noninvasive, extremely sensitive, and highly specific exam is critical, particularly for mucosal leishmaniasis (ML), in which a low parasite quantity is expected. We aimed to compare the diagnostic accuracy of swab and biopsy sample analysis using SYBR Green- and TaqMan-based real-time PCR (qPCR) assays with that of a composite reference standard consisting of the Montenegro skin test, serology, histopathology, smears, culture, and conventional PCR. In total, 55 patients with ATL (ML, 18 patients; cutaneous leishmaniasis [CL], 37 patients) and 36 patients without ATL were studied. qPCR analysis of swabs was more accurate when using SYBR Green (87.88%; 95% confidence interval [CI], 77.86 to 93.73 patients) than when using TaqMan (78.79%; 95% CI, 67.49 to 86.92%) (P= 0.031). SYBR Green (84.72%; 95% CI, 74.68 to 91.25%) was also more accurate than TaqMan (73.61%; 95% CI, 62.42 to 82.41%) for biopsy samples (P= 0.008). All qPCR methods were 100% specific. Swabs and biopsy specimens had similar sensitivity when using the same chemistry (P= 0.125 for SYBR Green andP= 0.625 for TaqMan). Moreover, qPCR achieved better performance than most existing techniques used for the diagnosis of ATL and also detected theLeishmaniaparasite in a greater proportion of patients than the associated histopathology, smear, culture, and conventional PCR techniques did. Swabs therefore represent a useful diagnostic tool because they not only are noninvasive but also can achieve an accuracy similar to that of biopsy samples. The high accuracy of SYBR Green-based qPCR may also reduce the requirement for associated parasitological tests for ATL diagnosis.

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

Jurnal Sains Farmasi & Klinis ◽

10.29208/jsfk.2017.4.1.194 ◽

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.

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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

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638711425582 ◽

2011 ◽

Vol 23 (6) ◽

pp. 1160-1167 ◽

Cited By ~ 3

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.

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Comparison of Two DNA Extractions and Nested PCR, Real-Time PCR, a New Commercial PCR Assay, and Bacterial Culture for Detection of Mycobacterium Avium Subsp. Paratuberculosis in Bovine Feces

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063870301500201 ◽

2003 ◽

Vol 15 (2) ◽

pp. 87-93 ◽

Cited By ~ 23

Author(s):

Jane Christopher-Hennings ◽

Matthew A. Dammen ◽

Shelleen R. Weeks ◽

William B. Epperson ◽

Shri N. Singh ◽

...

Keyword(s):

Real Time ◽

Southern Blot ◽

Real Time Pcr ◽

Nested Pcr ◽

Bacterial Culture ◽

Pcr Assay ◽

Polymerase Chain ◽

Mycobacterium Avium Subsp Paratuberculosis ◽

Pcr Techniques ◽

Bovine Feces

In this study, 5 combinations of 2 DNA extractions and 3 polymerase chain reaction (PCR) techniques were compared with culture for the detection of Mycobacterium paratuberculosis directly from bovine feces. These combinations included a new commercial extraction technique combined with a commercial PCR/Southern blot technique, nested PCR (nPCR), or real-time PCR, and a university-developed extraction combined with nPCR or real-time PCR. Four of the 5 combinations had statistically similar sensitivities between 93% and 100% and specificity between 95% and 100%, when compared with culture results from 63 bovine fecal samples. These results indicated that using a commercial extraction with a commercial PCR/Southern blot, nPCR, or real-time PCR, or a university-developed extraction with real-time PCR would result in similar sensitivities to culture for the identification of M. paratuberculosis from bovine feces and are valid alternatives to culture.

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

The Scientific World JOURNAL ◽

10.1155/2014/645084 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 4

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.

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Isolation and identification of Marek’s disease virus (MDV) from feather follicle epithelium and internal organs of diseased chickens in Dakahlia Governorate, Egypt

Mansoura Veterinary Medical Journal ◽

10.35943/mvmj.2019.22.102 ◽

2019 ◽

Vol 20 (2) ◽

pp. 6-11

Author(s):

Aly El-Kenawy ◽

Mohamed El-Tholoth ◽

Emad A

Keyword(s):

Real Time ◽

Disease Virus ◽

Real Time Pcr ◽

Marek's Disease Virus ◽

Marek's Disease ◽

Marek’S Disease Virus ◽

Marek’S Disease ◽

Field Samples ◽

Feather Follicle ◽

Positive Results

In the present study, a total of 16 samples including feather follicle epithelium, ovary, spleen and kidney (4 samples for each organ) were collected from diseased chicken flocks suspected to be infected with Marek’s disease virus (MDV) at Dakahlia Governorate, Egypt during the period from October 2016 to October 2017. Each sample was pooled randomly from three to five birds (90 to 360 days old). The isolation of the suspected virus from the collected samples was carried out via chorioallantoic membranes (CAMs) of 12 days old embryonated chicken eggs (ECEs). Three egg passages were carried out for each sample. Hyperimmune serum was prepared against standard MDV. MDV in both field and egg passaged samples (after 3rd passage) was identified by agar gel precipitation test (AGPT) and indirect fluorescence antibody test (IFAT). Molecular identification of virus was carried out by conventional polymerase chain reaction (PCR) and real- time PCR in four selected samples. The results revealed that 14 samples (87.5%) including 4 (100%) samples from feather follicle epithelium, ovary and kidney and 2 (50%) samples from spleen, showed positive results in virus isolation after 3rd passage. The positive results percentage by AGPT for field samples were 50% (8 out of 16 samples), while after the 3rd passage in ECEs were 37.5% (6 out of 16 samples) and the positive results percentage by IFAT for field samples were 62.5% (10 out of 16 samples), while after the 3rd passage in ECEs were 81.25 % (13 out of 16 samples). Viral nucleic acid was detected in all selected samples by conventional and real- time PCR. The results indicate that feather follicle epithelium is the best organ for MDV detection. IFAT is superior over AGPT in virus detection. Conventional and real - time PCR could be efficiently used for molecular detection of the virus.

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