scholarly journals Evaluation of designed IS711 primers and universal primers of B4 and B5 for detection of Brucella spp. in clinical samples

2019 ◽  
Author(s):  
Pedram Heidari ◽  
Mitra Salehi ◽  
Abbas Akhavan Sepahi ◽  
Mohamad Reza Razavi
Keyword(s):  
Blood Culture ◽  
Universal Primers ◽  
Clinical Samples ◽  
Target Sequence ◽  
Serum Samples ◽  
Serological Tests ◽  
Molecular Approaches ◽  
Bacterial Agent ◽  
Detection And Identification ◽  
Wide Range

Abstract Background Brucellosis as a global concern is a zoonotic infectious disease which affects a wide range of individual in developing countries. A confirmed diagnosis is required to isolate the bacterial agent from clinical specimens like blood, bone marrow, CSF or tissues. Microbiological, serological and molecular approaches are useful for detection and identification of Brucella spp. and blood culture is known as the gold standard for Brucella spp. Diagnosis of brucellosis through polymerase chain reaction (PCR) could be more sensitive and specific than other classical methods such as blood culture and conventional serological tests. Until now different Brucella specific sequences like BCSP 31, IS711 and 16SrRNAwere amplified for detection of Brucella Spp. Results Amplification of extracted DNA from serum of 49 suspected patients were tested with two sets of specific primers. The BCSP31 sequence amplicon was 223 bp and all the 49 (100%) serum specimens isolated from suspected patients were positive by B4 and B5 primers, even the 4 cases out of 49 2ME negative samples were positive. Detection of Brucella in serum samples by designed IS711 primers revealed the amplicon of IS711 with 448 bp length. Among the 49 serum samples isolated from patients, 46 (93.87%) cases were positive. The B4-B5 primers and IS711 designed primer recognized 100% (49/49) and 94% (46/49) of the cases, respectively. Conclusion This study shows that the specificity of the 2 primer sets is 100% and the sensitivity of B4-B5 primers is 100%, while the sensitivity of the designed primers of IS711 is 94%. The B4-B5 primers can detect the least number of both B. melitensis and B. abortus, 0.05 CFU/reaction. However, the designed IS711 set is able to detect 2 CFU/reaction, about 2.5×102 times more sensitive than results of other experiments for detection of IS711 target sequence in the specimens.

scholarly journals Evaluation of designed IS711 primers and universal primers of B4 and B5 for detection of Brucella spp. in clinical samples

2020 ◽  
Author(s):  
Pedram Heidari ◽  
Mitra Salehi ◽  
Abbas Akhavan Sepahi ◽  
Mohamad Reza Razavi
Keyword(s):  
Diagnostic Techniques ◽  
The Other ◽  
Universal Primers ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Molecular Diagnostic Techniques ◽  
Target Sequence ◽  
Molecular Approaches ◽  
Detection And Identification ◽  
Antibody Titers

Abstract Background: Brucellosis as a global concern is a zoonotic infectious disease which affects a large number of individuals in developing countries. Microbiological, serological and molecular approaches are useful for detection and identification of Brucella spp. A confirmed diagnosis requires isolation of Brucella from clinical specimens that is the most sensitive method in the acute and sub-acute phases of the diseases. On the other hand, molecular diagnostic techniques are more sensitive and more specific than serological techniques, especially in chronic localized cases because of antigenic cross-reactions or antibody titers lower than 160. Until now different Brucella specific sequences like BCSP 31, IS711 and 16SrRNA have been amplified for detection of Brucella spp. In this study, the sensitivity and specificity of The B4-B5 primers and IS711 designed primers were evaluated for detection of of Brucella Spp. in the clinical samples. Results : Amplification of extracted DNA from serum of 49 suspected patients were tested with two sets of specific primers. The BCSP31 amplicon was 223 bp and all the 49 (100%) serum specimens were positive by B4-B5 primers, including 4 cases with negative 2ME test result. The designed IS711 primers amplified the IS711 product with 448 bp length and 46 of 49 (93.87%) cases were positive. The sensitivity of the applied primers (B4-B5 and IS711) was evaluated by using the serial dilutions of extracted purified DNA molecules of B. melitensis and B. abortus . The B4-B5 primers can detect the least number of both B. melitensis and B. abortus , 0.1 CFU/reaction. However, the designed IS711 set is able to detect 10 CFU/reaction. The B4-B5 primer and IS711 designed primer recognized 100% (49/49) and 94% (46/49) of the cases, respectively. Conclusion: This study indicated that the sensitivity of B4-B5 primer is 100%, while the sensitivity of the designed primer of IS711 is 94%. The laboratory experiment revealed that designed IS711 set is 1×10 2 times more sensitive than sensitivity of the other experiments for detection of IS711 target sequence in the specimens.

scholarly journals Real-time PCR for detecting circulating dengue virus in the Guangdong Province of China in 2006

2008 ◽  
Vol 57 (12) ◽  
pp. 1547-1552 ◽  
Author(s):  
Zhijun Bai ◽  
Licheng Liu ◽  
Zeng Tu ◽  
Lisi Yao ◽  
Jianwei Liu ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Real Time ◽  
Real Time Pcr ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Sequencing Analysis ◽  
Serum Samples ◽  
Serological Tests ◽  
Wide Range ◽  
Pcr Assays

Dengue virus (DENV) causes a wide range of diseases in humans, from the acute febrile illness dengue fever (DF) to life-threatening dengue haemorrhagic fever/dengue shock syndrome. We developed four real-time quantitative PCR assays for each serotype of DENV based on computational analysis. These assays had high sensitivity and specificity without cross-reactivity for the four serotypes. To evaluate the performance of these assays in detecting and typing the virus in clinical samples, we analysed 64 serum samples from Guangdong during 2006. The results showed that 71 % of those samples were positive by the DEN-1 assay. The DENV assay results, in agreement with the serological tests and sequencing analysis, showed that the pathogen resulting in the DF explosion in Guangdong in 2006 belonged to DEN-1. Compared to the serological assays, the real-time PCR assays that we developed were much more sensitive in the 1–3 days after onset of the symptoms.

scholarly journals Clinical guideline for diagnosis and management of melioidosis

2006 ◽  
Vol 48 (1) ◽  
pp. 1-4 ◽  
Author(s):  
Timothy J.J. Inglis ◽  
Dionne B. Rolim ◽  
Jorge L.N. Rodriguez
Keyword(s):  
Oral Treatment ◽  
Central Nervous System Infection ◽  
Severe Infection ◽  
Community Acquired Pneumonia ◽  
Clinical Samples ◽  
Clinical Microbiology Laboratory ◽  
Serological Tests ◽  
Wide Range ◽  
Severe Community Acquired Pneumonia ◽  
Diagnostic Microbiology

Melioidosis is an emerging infection in Brazil and neighbouring South American countries. The wide range of clinical presentations include severe community-acquired pneumonia, septicaemia, central nervous system infection and less severe soft tissue infection. Diagnosis depends heavily on the clinical microbiology laboratory for culture. Burkholderia pseudomallei, the bacterial cause of melioidosis, is easily cultured from blood, sputum and other clinical samples. However, B. pseudomallei can be difficult to identify reliably, and can be confused with closely related bacteria, some of which may be dismissed as insignificant culture contaminants. Serological tests can help to support a diagnosis of melioidosis, but by themselves do not provide a definitive diagnosis. The use of a laboratory discovery pathway can help reduce the risk of missing atypical B. pseudomallei isolates. Recommended antibiotic treatment for severe infection is either intravenous Ceftazidime or Meropenem for several weeks, followed by up to 20 weeks oral treatment with a combination of trimethoprim-sulphamethoxazole and doxycycline. Consistent use of diagnostic microbiology to confirm the diagnosis, and rigorous treatment of severe infection with the correct antibiotics in two stages; acute and eradication, will contribute to a reduction in mortality from melioidosis.

scholarly journals Exploratory and confirmatory molecular approaches to determine genetically modified status in different crops

2021 ◽  
Vol 45 (1) ◽  
Author(s):  
Hanaa Abdel-Sadek Oraby ◽  
Nadia Aboul-Ftooh Aboul-Maaty ◽  
Hayam Ahmad Al-Sharawi
Keyword(s):  
Genetically Modified Organisms ◽  
Genetically Modified ◽  
Melting Curve ◽  
Conventional Polymerase Chain Reaction ◽  
Melting Curve Analysis ◽  
Target Sequence ◽  
Molecular Approaches ◽  
Detection And Identification ◽  
Food And Feed ◽  
Detection And Quantification

Abstract Background One of the parameters required for the assessment of food and feed safety is detection and identification of genetically modified organisms. Legislation in some countries necessitates detection and quantification of modification in food and feed samples. Scientists have raised concern about safety of antibiotic resistance marker (ARM) genes used for transformation of crops intended for human and animal consumption. In the present work two molecular approaches have been adopted: one exploratory; for detection and quantification of ARM genes in tested plant samples and the other confirmatory; to determine the specificity/reliability of the obtained results. Results Results revealed that primers for neomycin phosphotransferase (nptII) and aminoglycoside 3″ adenyl-transferase (aadA) were amplified in the majority of the 36 DNA screened samples. Melting curve analysis using hygromycin phosphotransferase (aphIV) gene as target sequence for the fluorescent-based detection approach was performed to ensure reliability and specificity of this procedure and to confirm results obtained by using conventional polymerase chain reaction (PCR). Quantitative RT-PCR results and validation analysis followed, revealed that all of the tested DNA samples were not violating the European legislation for GMOs labeling (0.9%). Conclusions The results fully demonstrated the reproducibility, sensitivity/specificity of the adopted approaches for detection and quantification of even traces of GMO contents. Applying measurement uncertainty (MU) procedures presented in this work will help decision makers to ensure compliance with International Legislation and Regulations. This in its turn will facilitate and enhance trading with countries having compelling labeling regulations.

scholarly journals A Half-Day Genome Sequencing Protocol for Middle East Respiratory Syndrome Coronavirus

2021 ◽  
Vol 12 ◽  
Author(s):  
Kwan Woo Kim ◽  
Sungmi Choi ◽  
Su-Kyoung Shin ◽  
Imchang Lee ◽  
Keun Bon Ku ◽  
...  
Keyword(s):  
Public Health ◽  
Middle East ◽  
Genome Sequencing ◽  
Complete Genome ◽  
Middle East Respiratory Syndrome ◽  
Universal Primers ◽  
Clinical Samples ◽  
Primary Concern ◽  
Whole Genome ◽  
Serum Samples

Recent coronavirus (CoV) outbreaks, including that of Middle East respiratory syndrome (MERS), have presented a threat to public health worldwide. A primary concern in these outbreaks is the extent of mutations in the CoV, and the content of viral variation that can be determined only by whole genome sequencing (WGS). We aimed to develop a time efficient WGS protocol, using universal primers spanning the entire MERS-CoV genome. MERS and synthetic Neoromicia capensis bat CoV genomes were successfully amplified using our developed PCR primer set and sequenced with MinION. All experimental and analytical processes took 6 h to complete and were also applied to synthetic animal serum samples, wherein the MERS-CoV genome sequence was completely recovered. Results showed that the complete genome of MERS-CoV and related variants could be directly obtained from clinical samples within half a day. Consequently, this method will contribute to rapid MERS diagnosis, particularly in future CoV epidemics.

scholarly journals Next-Generation Sequencing to Detect Pathogens in Pediatric Febrile Neutropenia: A Single-Center Retrospective Study of 112 Cases

2021 ◽  
Author(s):  
Kazuhiro Horiba ◽  
Yuka Torii ◽  
Toshihiko Okumura ◽  
Suguru Takeuchi ◽  
Takako Suzuki ◽  
...  
Keyword(s):  
Cluster Analysis ◽  
Next Generation Sequencing ◽  
Blood Culture ◽  
Febrile Neutropenia ◽  
Clinical Samples ◽  
Next Generation ◽  
Serum Samples ◽  
Blood Samples ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Background Febrile neutropenia (FN) is a frequent complication in immunocompromised patients. However, causative microorganisms are detected in only 10% of patients. This study aimed to detect the microorganisms that cause FN using next-generation sequencing (NGS) to idenjpgy the genome derived from pathogenic microorganisms in the bloodstream. Here, we implemented a metagenomic approach to comprehensively analyze microorganisms present in clinical samples from patients with FN. Methods FN is defined as 1) a neutrophil count < 500/µL, and 2) fever ≥ 37.5 °C. Plasma/serum samples of 112 pediatric patients with FN, 10 patients with neutropenia without fever (NE), were sequenced by NGS and analyzed by a metagenomic pipeline PATHDET. Results The putative pathogens were detected by NGS in 5 of 10 patients with FN with positive for blood culture results, 15 of 87 patients (17%) with negative for blood culture results, and 3 of 8 patients with NE. Several bacteria that were common in the oral, skin, and gut flora were commonly detected in blood samples, suggesting translocation of the human microbiota to the bloodstream in the setting of neutropenia. The cluster analysis of the microbiota in blood samples using NGS demonstrated that the representative bacteria of each cluster was mostly consistent with the pathogens in each patient. Conclusions NGS technique has a great potential for detecting causative pathogens in patients with FN. Cluster analysis, which extracts characteristic microorganisms from a complex microbial population, may be effective to detect pathogens in minute quantities of microbiota, such as those from the bloodstream.

scholarly journals Evaluation of nine commercial SARS-CoV-2 immunoassays

2020 ◽  
Author(s):  
Ria Lassaunière ◽  
Anders Frische ◽  
Zitta B. Harboe ◽  
Alex C.Y. Nielsen ◽  
Anders Fomsgaard ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Rank Order ◽  
Point Of Care ◽  
Epidemiological Studies ◽  
Contact Tracing ◽  
Clinical Samples ◽  
Serum Samples ◽  
Serological Tests ◽  
Barr Virus ◽  
Tract Infections

AbstractDue to urgency and demand, numerous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoassays are rapidly being developed and placed on the market with limited validation on clinical samples. Thorough validation of serological tests are required to facilitate their use in the accurate diagnosis of SARS-CoV-2 infection, confirmation of molecular results, contact tracing, and epidemiological studies. This study evaluated the sensitivity and specificity of nine commercially available serological tests. These included three enzyme-linked immunosorbent assays (ELISAs) and six point-of-care (POC) lateral flow tests. The assays were validated using serum samples from: i) SARS-CoV-2 PCR-positive patients with a documented first day of disease; ii) archived sera obtained from healthy individuals before the emergence of SARS-CoV-2 in China; iii) sera from patients with acute viral respiratory tract infections caused by other coronaviruses or non-coronaviruses; and iv) sera from patients positive for dengue virus, cytomegalovirus and Epstein Barr virus. The results showed 100% specificity for the Wantai SARS-CoV-2 Total Antibody ELISA, 93% for the Euroimmun IgA ELISA, and 96% for the Euroimmun IgG ELISA with sensitivities of 90%, 90%, and 65%, respectively. The overall performance of the POC tests according to manufacturer were in the rank order of AutoBio Diagnostics > Dynamiker Biotechnology = CTK Biotech > Artron Laboratories > Acro Biotech ≥ Hangzhou Alltest Biotech. Overall, these findings will facilitate selection of serological assays for the detection SARS-CoV-2-specific antibodies towards diagnosis as well as sero-epidemiological and vaccine development studies.

scholarly journals Comparison of Different PCR Methods for Detection of Brucella spp. in Human Blood Samples

2011 ◽  
Vol 60 (1) ◽  
pp. 27-33 ◽  
Author(s):  
HISHAM H. AL-AJLAN ◽  
ABDELNASSER S.S. IBRAHIM ◽  
ALI A. AL-SALAMAH
Keyword(s):  
Blood Culture ◽  
Real Time ◽  
Real Time Pcr ◽  
Brucella Melitensis ◽  
Blood Analysis ◽  
Clinical Samples ◽  
Conventional Pcr ◽  
Serum Samples ◽  
Culture Methods ◽  
Blood Specimens

For detection of Brucella species by PCR four DNA extraction methods and four targets were compared using pure culture of Brucella melitensis and the best conditions were applied in clinical samples. It was found that the MagNA Pure LC method was the most efficient and sensitive method showing a positive PCR reaction with DNA extracted from as low as 25 and 100 CFU suspended in one ml blood and one ml water, respectively. Detection of Brucella spp. by conventional PCR was investigated using four different targets. The results indicated that The B4-B5 amplification method was the most sensitive one as it could amplify DNA extracted from as a low as 25 and 100 CFU/ml suspended in one ml water and blood, respectively. Furthermore real-time PCR was able to detect Brucella using DNA extracted from as low as 50 CFU/ml blood and 15 CFU/ml water, respectively. The best and optimum detection conditions were applied to the clinical samples. Evaluation of conventional PCR assays on blood specimens confirmed 72% of the results obtained by conventional blood culture methods with a specificity of 95%, while serum samples had a sensitivity of 54% and specificity of 100%. Real-time PCR was generally found to be more sensitive and specific for detecting Brucella spp. in blood and serum samples compared to conventional PCR. The real-time PCR done on blood specimens confirmed 77.5% of the results obtained by conventional blood culture methods with specificity of 100%, while 60% of serum samples were found to be positive with specificity of 100%. These results suggest that serum and blood analysis by conventional and real time PCR is a convenient and safe method for rapid and accurate diagnosis of brucellosis.

scholarly journals Multicenter Evaluation of the Elecsys Hepatitis B Surface Antigen Quantitative Assay

2011 ◽  
Vol 18 (11) ◽  
pp. 1943-1950 ◽  
Author(s):  
B. J. Zacher ◽  
F. Moriconi ◽  
S. Bowden ◽  
R. Hammond ◽  
S. Louisirirotchanakul ◽  
...  
Keyword(s):  
Hepatitis B ◽  
Surface Antigen ◽  
Hepatitis B Surface Antigen ◽  
World Health ◽  
Quantitative Assay ◽  
Clinical Samples ◽  
High Dose ◽  
Serum Samples ◽  
Hook Effect ◽  
Wide Range

ABSTRACTThe Elecsys hepatitis B surface antigen (HBsAg) II quantitative assay is a new quantitative electrochemiluminescence immunoassay which uses onboard dilution and a simple algorithm to determine HBsAg levels expressed in international units (IU)/ml (standardized against the World Health Organization [WHO] Second International Standard). This study evaluated its performance using routine serum samples from a wide range of HBsAg carriers and patients with chronic hepatitis B (CHB). HBsAg levels were measured in serum samples collected independently by five centers in Europe, Australia, and Asia. Serial dilution analyses were performed to assess the recommended dilution algorithm and determine the assay range free of hook effect. Assay precision was also established. Following assessment of serial dilutions (1:100 to 1:1,000,000) of the 611 samples analyzed, 70.0% and 85.6% of samples tested with analyzers incorporating 1:100 (Elecsys 2010 and cobas e 411) and 1:400 (Modular Analytics E170) onboard dilution, respectively, fell within the linear range of the assay, providing a final result on the first test. No high-dose hook effect was seen up to the maximum HBsAg serum level tested (870,000 IU/ml) using the dilution algorithm. HBsAg levels were reliably determined across all hepatitis B virus (HBV) genotypes, phases of HBV infection, and stages of disease tested. Precision was high across all analyzers (% coefficient of variation [CV], 1.4 to 9.6; HBsAg concentrations, 0.1 to 37,300 IU/ml). The Elecsys HBsAg II quantitative assay accurately and reliably quantifies HBsAg in routine clinical samples. Onboard dilution minimizes retesting and reduces the potential for error.

Sandwich dot-immunogold filtration assay (DIGFA) for specific immunodiagnosis of active neuroangiostrongyliasis

Parasitology ◽  
2020 ◽  
pp. 1-6
Author(s):  
Praphathip Eamsobhana ◽  
Anchalee Tungtrongchitr ◽  
Hoi-Sen Yong ◽  
Anchana Prasartvit ◽  
Darawan Wanachiwanawin ◽  
...  
Keyword(s):  
False Negative ◽  
Clinical Samples ◽  
Serum Samples ◽  
Serological Tests ◽  
Past Infection ◽  
Negative Results ◽  
Resource Limited ◽  
False Negative Results ◽  
Circulating Antigens ◽  
Specific Antigens

Abstract Serological tests may yield false-negative results for specific antibodies detection before or at the early seroconversion phase. Tests that detect circulating antigens of Angiostrongylus cantonensis would therefore be of value in diagnosis to distinguish current or past infection. Here, a quick, easy to perform, portable and inexpensive diagnostic device for detection of 31-kDa A. cantonensis specific antigens had been developed. This sandwich dot-immunogold filtration assay (AcDIGFAAg), for detecting active angiostrongyliasis was produced using anti-A. cantonensis polyclonal antibody dotted on the nitrocellulose membrane as a capture agent and colloidal gold-labelled anti-31 kDa A. cantonensis antibody as a detection agent. A well-defined pink dot, indicating positivity, was seen readily by naked eye within 10–15 min. The AcDIGFAAg detected A. cantonensis-specific antigens in cerebrospinal fluid samples from 4 out of 10 serologically confirmed angiostrongyliasis cases and 2 out of 5 suspected cases with negative anti-A. cantonensis antibodies. Among the 19 patient sera with A. cantonensis infection, 2 showed positive reaction by AcDIGFAAg. No positive AcDIGFAAg reaction was observed in all the serum samples with other parasitic diseases, and the healthy controls. The present ‘AcDIGFAAg’ enables rapid qualitative detection of the specific 31-kDa antigens of A. cantonensis in clinical samples with potential for application even under resource-limited settings.

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