scholarly journals Application of Fluorescence Based Molecular Assays for Improved Detection and Typing of Brucella Strains in Clinical Samples

2015 ◽  
Vol 38 (2) ◽  
pp. 223-232 ◽  
Author(s):  
Kiril Krstevski ◽  
Ivancho Naletoski ◽  
Dine Mitrov ◽  
Slavcho Mrenoshki ◽  
Iskra Cvetkovikj ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic Algorithm ◽  
Diagnostic Procedures ◽  
Molecular Techniques ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Tissue Samples ◽  
Study Results ◽  
Epidemiological Situation

AbstractBacteria from the genus Brucella are causative agents of brucellosis - a zoonotic disease which affects many wild and domestic animal species and humans. Taking into account the significant socio-economic and public health impact of brucellosis, its control is of great importance for endemic areas. The chosen control strategy could be successful only if adapted to the current epidemiological situation. This implies that a choice of appropriate diagnostic procedures for detection and typing of Brucella spp. strains are of essential importance. Significant advancement of molecular techniques and their advantages compared to classical methods, give strong arguments in promotion of these techniques as a powerful tool for comprehensive diagnostics of brucellosis. Considering this, the major tasks of the study were to select and implement molecular tests for detection and genotyping Brucella spp. and evaluate their performances using DNA from cultivated brucellae (islolates) and limited number of tissue samples from seropositive animals. The obtained results confirmed that implemented real time PCR for Brucella spp. detection, as well as MLVA-16 used for genotyping, have excellent analytical sensitivity (4.2 fg of Brucella DNA were successfully detected and genotyped). Furthermore, compared to bacteriological cultivation of Brucella spp., real time PCR and MLVA-16 protocols showed superior diagnostic sensitivity and detected Brucella DNA in tissues from which Brucella could not be cultivated. Based on the summarized study results, we propose a diagnostic algorithm for detection and genotyping of Brucella spp. bacteria. Routine use of proposed diagnostic algorithm will improve the effectiveness of infection confirmation and help for accurate evaluation of epidemiological situation.

scholarly journals Comparative evaluation of published cytomegalovirus primers for rapid real-time PCR: which are the most sensitive?

2009 ◽  
Vol 58 (7) ◽  
pp. 878-883 ◽  
Author(s):  
Wafa Habbal ◽  
Fawza Monem ◽  
Barbara C. Gärtner
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Clinical Isolates ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Pcr Product ◽  
Highly Sensitive ◽  
Primer Sets ◽  
Analytical Sensitivities ◽  
Strain Ad169

Standardization of human cytomegalovirus (CMV) PCR is highly recommended. As primer design is essential for PCR sensitivity, this study evaluated all published CMV primer pairs to identify the most sensitive for single-round real-time PCR. PubMed (1993–2004) was searched for original papers aimed at CMV PCR. Fifty-seven papers were identified revealing 82 different primer pairs. Of these, 17 primer sets were selected for empirical study, as they were either used in real-time PCR or were evaluated comparatively by conventional PCR. After optimizing the PCR conditions, these primer sets were evaluated by real-time PCR using a SYBR Green format. Analytical sensitivities were assessed by testing the reference standard CMV strain AD169. A blast search was performed to identify mismatches with published sequences. Additionally, 60 clinical samples were tested with the three primer sets showing highest analytical sensitivity and the best match to all CMV strains. Three primer sets located in the glycoprotein B (UL55) gene region were found to be the most sensitive using strain AD169. However, two of these showed a considerable number of mismatches with clinical isolates in a blast search. Instead, two other pairs from the lower matrix phosphoprotein (UL83) gene and DNA polymerase (UL54) gene showed reasonable sensitivity and no mismatches with clinical isolates. These three pairs were further tested with clinical samples, which indicated that the two primer sets from UL55 and UL54 were the most sensitive. Interestingly, the analytical sensitivity of the PCR was inversely correlated with the size of the PCR product. In conclusion, these two primer pairs are recommended for a standardized, highly sensitive, real-time PCR.

Investigation of brucellosis in cattle and buffaloes by conventional and molecular assays

2017 ◽  
Author(s):  
Paviter Kaur ◽  
N. S. Sharma ◽  
A. K. Arora ◽  
Deepti .
Keyword(s):  
Multiplex Pcr ◽  
Real Time ◽  
Brucella Abortus ◽  
Real Time Pcr ◽  
Stomach Contents ◽  
Molecular Techniques ◽  
Clinical Samples ◽  
Bovine Brucellosis ◽  
Molecular Assays

The present study was carried out to evaluate the conventional and molecular techniques for diagnosis of bovine brucellosis. A total of four isolates of Brucella abortus obtained from 100 clinical samples of foetal stomach contents, vaginal mucus and uterine discharges were characterized biochemically. The isolates were confirmed as Brucella spp. by PCR using B4/B5 primer pair and as B. abortus by Bruce Ladder multiplex PCR. By Hinic Real-time PCR, all the four isolates were confirmed as Brucella spp. with Ct values between 14-16. With DNA extracted from 40 clinical samples of foetal stomach contents, vaginal mucus and uterine discharges, Real time PCR appeared most sensitive of the three molecular and conventional techniques as it detected maximum number of positive samples.

scholarly journals Development and evaluation of a multiplex loop-mediated isothermal amplification (LAMP) assay for differentiation of Mycobacterium tuberculosis and non-tuberculosis mycobacterium in clinical samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0244753
Author(s):  
Jeeyong Kim ◽  
Borae G. Park ◽  
Da Hye Lim ◽  
Woong Sik Jang ◽  
Jeonghun Nam ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Positive Reaction ◽  
Real Time Pcr ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Published Data ◽  
Analytical Sensitivity ◽  
Loop Mediated Isothermal Amplification

Introduction The rapid and accurate diagnosis of tuberculosis (TB) is important to reduce morbidity and mortality rates and risk of transmission. Therefore, molecular detection methods such as a real-time PCR–based assay for Mycobacterium tuberculosis (MTB) have been commonly used for diagnosis of TB. Loop-mediated isothermal amplification (LAMP) assay was believed to be a simple, quick, and cost-effective isothermal nucleic acid amplification diagnostic test for infectious diseases. In this study, we designed an in-house multiplex LAMP assay for the differential detection of MTB and non-tuberculosis mycobacterium (NTM), and evaluated the assay using clinical samples. Material and methods For the multiplex LAMP assay, two sets of specific primers were designed: the first one was specific for IS6110 genes of MTB, and the second one was universal for rpoB genes of mycobacterium species including NTM. MTB was confirmed with a positive reaction with both primer sets, and NTM was identified with a positive reaction by only the second primer set without a MTB-specific reaction. Total 333 clinical samples were analyzed to evaluate the multiplex LAMP assay. Clinical samples were composed of 195 positive samples (72 MTB and 123NTM) and 138 negative samples. All samples were confirmed positivity or negativity by real-time PCR for MTB and NTM. Analytical sensitivity and specificity were evaluated for the multiplex LAMP assay in comparison with acid fast bacilli staining and the culture method. Results Of 123 NTM samples, 121 were identified as NTM and 72/72 MTB were identified as MTB by the multiplex LAMP assay. False negative reactions were seen only in two NTM positive samples with co-infection of Candida spp. All 138 negative samples were identified as negative for MTB and NTM. Analytical sensitivity of the multiplex LAMP assay was 100% (72/72) for MTB, and 98.4% (121/123) for NTM. And the specificity of assay was 100% (138/138) for all. Conclusions Our newly designed multiplex LAMP assay for MTB and NTM showed relatively good sensitivity in comparison with previously published data to detect isolated MTB. This multiplex LAMP assay is expected to become a useful tool for detecting and differentiating MTB from NTM rapidly at an acceptable sensitivity.

scholarly journals Rapid Detection and Differentiating of the Predominant Salmonella Serovars in Chicken Farm by TaqMan Multiplex Real-Time PCR Assay

2021 ◽  
Vol 11 ◽  
Author(s):  
Suhua Xin ◽  
Hong Zhu ◽  
Chenglin Tao ◽  
Beibei Zhang ◽  
Lan Yao ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Epidemiologic Study ◽  
Clinical Diagnostics ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Chicken Farm

Salmonella has been known as an important zoonotic pathogen that can cause a variety of diseases in both animals and humans. Poultry are the main reservoir for the Salmonella serovars Salmonella Pullorum (S. Pullorum), Salmonella Gallinarum (S. Gallinarum), Salmonella Enteritidis (S. Enteritidis), and Salmonella Typhimurium (S. Typhimurium). The conventional serotyping methods for differentiating Salmonella serovars are complicated, time-consuming, laborious, and expensive; therefore, rapid and accurate molecular diagnostic methods are needed for effective detection and prevention of contamination. This study developed and evaluated a TaqMan multiplex real-time PCR assay for simultaneous detection and differentiation of the S. Pullorum, S. Gallinarum, S. Enteritidis, and S. Typhimurium. In results, the optimized multiplex real-time PCR assay was highly specific and reliable for all four target genes. The analytical sensitivity corresponded to three colony-forming units (CFUs) for these four Salmonella serovars, respectively. The detection limit for the multiplex real-time PCR assay in artificially contaminated samples was 500 CFU/g without enrichment, while 10 CFU/g after pre-enrichment. Moreover, the multiplex real-time PCR was applied to the poultry clinical samples, which achieved comparable results to the traditional bacteriological examination. Taken together, these results indicated that the optimized TaqMan multiplex real-time PCR assay will be a promising tool for clinical diagnostics and epidemiologic study of Salmonella in chicken farm and poultry products.

scholarly journals Development of a real-time PCR assay for the detection of Brucella ovis DNA in clinical samples

2021 ◽  
Vol 7 (1-2) ◽  
pp. 17-20
Author(s):  
N. V. Marchenko ◽  
O. Yu. Lymanska ◽  
A. P. Gerilovych ◽  
V. I. Bolotin
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Closely Related Species ◽  
Tissue Samples ◽  
Fast Detection ◽  
Brucella Ovis ◽  
Direct Indication ◽  
Pcr Targeting

The etiological agent of infectious ovine epididymitis is Brucella ovis and for its direct indication in clinical samples several PCR protocols are proposed. This study describes a design and selection of the oligonucleotides for real-time PCR targeting conservative BOV_A0504 gene. The specificity of a real-time PCR was validated using 25 B. ovis field isolates and 14 microorganisms of closely related species. The detection limit of B. ovis in bacterial culture was determined as 3.5×101 CFU/mL with Ct value of 37.8. There are no detectable fluorescence signals in the clinical samples from intact animals, whereas bacteriologically confirmed material such as urine and testicle tissue samples were positive. It confirms that the assay is highly specific for detection of B. ovis DNA. Thus, the proposed real-time PCR assay enables fast detection and quantification of B. ovis in clinical material, which can be used as additional test for estimation of the health status of a sheep herd

scholarly journals Validation of Nonnested and Real-Time PCR for Diagnosis of Sheep-Associated Malignant Catarrhal Fever in Clinical Samples

2007 ◽  
Vol 19 (4) ◽  
pp. 405-408 ◽  
Author(s):  
Donald L. Traul ◽  
Naomi S. Taus ◽  
J. Lindsay Oaks ◽  
Donal O' Toole ◽  
Fred R. Rurangirwa ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
High Specificity ◽  
Clinical Samples ◽  
Malignant Catarrhal Fever ◽  
Fatal Disease ◽  
Tissue Samples ◽  
Specificity And Sensitivity ◽  
Experimentally Induced

Sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal disease primarily of certain ruminants, is caused by ovine herpesvirus 2 (OvHV-2). Molecular diagnosis of SA-MCF in affected animals has relied on detection of OvHV-2 DNA using a nested PCR, which has significant potential for amplicon contamination as a routine method in diagnostic laboratories. In this report, a nonnested and a previously developed real-time PCR were validated for detection of OvHV-2 DNA in samples from clinically affected animals. Three sets of blood or tissue samples were collected: 1) 97 samples from 97 naturally affected animals with evidence of clinical SA-MCF; 2) 200 samples from 8 animals with experimentally induced SA-MCF; and 3) 100 samples from 100 animals without any evidence of clinical SA-MCF. Among 97 positive samples defined by nested PCR from clinically affected animals, 95 (98%) were positive by nonnested PCR and 93 (96%) were positive by real-time PCR, respectively. One hundred percent of the samples from the animals with experimentally induced MCF were positive by real-time PCR, while 99% were positive by nonnested PCR. Neither nonnested PCR nor real-time PCR yielded a positive result on any of the 100 nested PCR-negative samples from animals without evidence of clinical MCF. The data confirmed that both nonnested and real-time PCR maintained high specificity and sensitivity for the detection of OvHV-2 DNA in clinical samples.

HybProbes-based real-time PCR assay for rapid detection of equine herpesvirus type 2 DNA

2012 ◽  
Vol 15 (3) ◽  
pp. 411-416 ◽  
Author(s):  
E. Osińska ◽  
A. Golke ◽  
A. Słońska ◽  
J. Cymerys ◽  
M.W. Bańbura ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Viral Dna ◽  
Herpesvirus Type ◽  
Equid Herpesvirus

Abstract Equid herpesvirus type 2 (EHV-2) together with equid herpesvirus type 5 are members of Gammaherpesvirinae subfamily, genus Rhadinovirus. EHV-2 is one of major agents causing diseases of horses common worldwide. A possible role of EHV-2 in reactivating latent equid herpesvirus type-1 has been suggested, because reactivation of latent EHV-1 was always accompanied by EHV-2 replication. Variety techniques, including cell culture, PCR and its modifications, have been used to diagnose EHV-2 infections. The aim of this study was to develop, optimize and determine specificity of real-time PCR (qPCR) for EHV-2 DNA detection using HybProbesR chemistry and to evaluate clinical samples with this method. The analytical sensitivity of assay was tested using serial dilutions of viral DNA in range between 70 and 7x105 copies/ml. The limit of detection (LOD) was calculated using probit analysis and was determined as 56 copies/ml. In further studies 20 different clinical samples were tested for the presence of EHV-2. Described in-house qPCR method detected viral DNA in 5 of 20 specimens used. The results of this work show that developed HybProbes-based real-time PCR assay is very reliable and valuable for detection and quantification of equid herpesvirus type 2 DNA in different clinical samples. The high level of sensitivity, accuracy and rapidity provided by the LightCycler 2.0 instrument are favorable for the use of this system in the detection of EHV-2 DNA in veterinary virology.

scholarly journals Validation of a Novel Diagnostic Approach Combining the VersaTREK™ System for Recovery and Real-Time PCR for the Identification of Mycobacterium chimaera in Water Samples

2021 ◽  
Vol 9 (5) ◽  
pp. 1031
Author(s):  
Roberto Zoccola ◽  
Alessia Di Blasio ◽  
Tiziana Bossotto ◽  
Angela Pontei ◽  
Maria Angelillo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Detection System ◽  
Bacterial Load ◽  
Microbial Control ◽  
Hospital Setting ◽  
Sample Volume ◽  
Analytical Sensitivity ◽  
Initial Water

Mycobacterium chimaera is an emerging pathogen associated with endocarditis and vasculitis following cardiac surgery. Although it can take up to 6–8 weeks to culture on selective solid media, culture-based detection remains the gold standard for diagnosis, so more rapid methods are urgently needed. For the present study, we processed environmental M. chimaera infected simulates at volumes defined in international guidelines. Each preparation underwent real-time PCR; inoculates were placed in a VersaTREK™ automated microbial detection system and onto selective Middlebrook 7H11 agar plates. The validation tests showed that real-time PCR detected DNA up to a concentration of 10 ng/µL. A comparison of the isolation tests showed that the PCR method detected DNA in a dilution of ×102 CFU/mL in the bacterial suspensions, whereas the limit of detection in the VersaTREK™ was <10 CFU/mL. Within less than 3 days, the VersaTREK™ detected an initial bacterial load of 100 CFU. The detection limit did not seem to be influenced by NaOH decontamination or the initial water sample volume; analytical sensitivity was 1.5 × 102 CFU/mL; positivity was determined in under 15 days. VersaTREK™ can expedite mycobacterial growth in a culture. When combined with PCR, it can increase the overall recovery of mycobacteria in environmental samples, making it potentially applicable for microbial control in the hospital setting and also in environments with low levels of contamination by viable mycobacteria.

scholarly journals Real-time PCR strategy for parasite quantification in blood and tissue samples of experimental Trypanosoma cruzi infection

Acta Tropica ◽  
2012 ◽  
Vol 123 (3) ◽  
pp. 170-177 ◽  
Author(s):  
Sérgio Caldas ◽  
Ivo Santana Caldas ◽  
Lívia de Figueiredo Diniz ◽  
Wanderson Geraldo de Lima ◽  
Riva de Paula Oliveira ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Real Time ◽  
Real Time Pcr ◽  
Tissue Samples ◽  
Cruzi Infection
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