scholarly journals Development of a technique for DNA detection and identification of toxigenic strains of Clostridium botulinum types A, B, E by the Real-Time PCR method

2018 ◽  
Vol 2 (4) ◽  
pp. 36-43
Keyword(s):  
Botulinum Toxin ◽  
Real Time ◽  
Clostridium Botulinum ◽  
Specific Activity ◽  
Food Samples ◽  
Analytical Sensitivity ◽  
Detection And Identification ◽  
The Central Nervous System ◽  
Toxigenic Strains ◽  
Definition Of

Botulism is dangerous toxic infection caused by a toxin produced by the bacterium Clostridium botulinum. The mortality rate from botulism can reach 70% of all cases of illness in case of untimely initiation of treatment. The pathogenesis of botulism involves the damage to the central nervous system by a toxin produced by C. botulinum. Currently there are seven recognized antigenic types of this toxin. Botulinum toxin is included into the group of biological agents and it is one of the most likely agents to be used in a biological attack. Since botulinum neurotoxin is a complex nucleoprotein complex and the traces of DNA can be detected even in purified toxin preparations, we have elaborated a technique for detecting and identifying DNA of toxigenic strains of Clostridium botulinum types A, B, E, that cause human botulism in most cases. This technique is based on the the detection of residual amounts of this DNA in botulinum toxin using multiplex real-time polymerase chain reaction (PCR) assay with fluorescent hybridization detection. The main obstacle to development of a technique for the detection and identification of DNA of toxigenic strains is the high variability of the genes responsible for the synthesis of botulinum toxin. We have established a region of the gene with the lowest homology in all strains. This requirement is met by a fragment of the bont gene that encodes a light chain of a neurotoxin and is highly conserved in the strains of C. botulinum producing one type of toxin. The paper represents the results of the definition of analytical sensitivity and specific activity of the developed method. The specificity of the determination is 100%, the analytical sensitivity – 1×10 2 mc./ml. The method can be used to analyze food, samples of clinical materials and environmental samples suspected of being contaminated with toxigenic strains of C. botulinum

scholarly journals Quadruplex Real-Time PCR Assay for Detection and Identification of Vibrio cholerae O1 and O139 Strains and Determination of Their Toxigenic Potential

2009 ◽  
Vol 75 (22) ◽  
pp. 6981-6985 ◽  
Author(s):  
Jianwei Huang ◽  
Yumei Zhu ◽  
Huixin Wen ◽  
Jiafeng Zhang ◽  
Shijie Huang ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Toxin Gene ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Detection And Identification ◽  
Life Threatening ◽  
Toxigenic Strains

ABSTRACT Vibrio cholerae is a natural inhabitant of the aquatic environment. However, its toxigenic strains can cause potentially life-threatening diarrhea. A quadruplex real-time PCR assay targeting four genes, the cholera toxin gene (ctxA), the hemolysin gene (hlyA), O1-specific rfb, and O139-specific rfb, was developed for detection and differentiation of O1, O139, and non-O1, non-O139 strains and for prediction of their toxigenic potential. The specificity of the assay was 100% when tested against 70 strains of V. cholerae and 31 strains of non-V. cholerae organisms. The analytical sensitivity for detection of toxigenic V. cholerae O1 and O139 was 2 CFU per reaction with cells from pure culture. When the assay was tested with inoculated water from bullfrog feeding ponds, 10 CFU/ml could reliably be detected after culture for 3 h. The assay was more sensitive than the immunochromatographic assay and culture method when tested against 89 bullfrog samples and 68 water samples from bullfrog feeding ponds. The applicability of this assay was confirmed in a case study involving 15 bullfrog samples, from which two mixtures of nontoxigenic O1 and toxigenic non-O1/non-O139 strains were detected and differentiated. These data indicate that the quadruplex real-time PCR assay can both rapidly and accurately detect/identify V. cholerae and reliably predict the toxigenic potential of strains detected.

scholarly journals Development of the Multiplex Real-Time PCR for Marburg, Ebola, and Lassa Viruses Identification

2011 ◽  
pp. 64-67
Author(s):  
A. O. Sementsova ◽  
A. N. Shikov ◽  
V. A. Ternovoy ◽  
A. V. Vinokurova ◽  
N. E. Kostina ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Real Time ◽  
Real Time Pcr ◽  
Specific Activity ◽  
Fluorescent Detection ◽  
Differential Diagnostics ◽  
Analytical Sensitivity ◽  
Hemorrhagic Fevers

Presented are the data on the development and approbation of the method of Marburg, Ebola, and Lassa viruses identification based on real-time multiplex PCR with hybridization-fluorescent detection. This method is meant for the differential diagnostics of hemorrhagic fevers caused by these viruses. Displayed are the results of determination of multiplex PCR analytical sensitivity and specific activity.

scholarly journals Botulism in Brazil, 2000-2008: epidemiology, clinical findings and laboratorial diagnosis

2010 ◽  
Vol 52 (4) ◽  
pp. 183-186 ◽  
Author(s):  
Ruth Estela Gravato Rowlands ◽  
Christiane Asturiano Ristori ◽  
Giselle Ibette S. Lopez Lopes ◽  
Ana Maria Ramalho de Paula ◽  
Harumi Sakuma ◽  
...  
Keyword(s):  
Clostridium Botulinum ◽  
Type A ◽  
Food Samples ◽  
Clinical Findings ◽  
Control Measures ◽  
Clinical Samples ◽  
Mouse Bioassay ◽  
Detection And Identification ◽  
Food Borne ◽  
Type Ab

Botulism is a rare and potentially lethal illness caused by Clostridium botulinum neurotoxin. We describe the findings of a laboratorial investigation of 117 suspected cases of botulism reported to the surveillance system in Brazil from January 2000 to October 2008. Data on the number and type of samples analyzed, type of toxins identified, reporting of the number of botulism cases and transmission sources are discussed. A total of 193 clinical samples and 81 food samples were analyzed for detection and identification of the botulism neurotoxin. Among the clinical samples, 22 (11.4%) presented the toxin (nine type A, five type AB and eight with an unidentified type); in food samples, eight (9.9%) were positive for the toxin (five type A, one type AB and two with an unidentified type). Of the 38 cases of suspected botulism in Brazil, 27 were confirmed by a mouse bioassay. Laboratorial botulism diagnosis is an important procedure to elucidate cases, especially food-borne botulism, to confirm clinical diagnosis and to identify toxins in food, helping sanitary control measures.

scholarly journals Development and Validation of a One-Step Reverse Transcription Real-Time PCR Assay for Simultaneous Detection and Identification of Tomato Mottle Mosaic Virus and Tomato Brown Rugose Fruit Virus

2021 ◽  
Author(s):  
Antonio Tiberini ◽  
Ariana Manglli ◽  
Anna Taglienti ◽  
Ana Vučurović ◽  
Jakob Brodaric ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Real Time ◽  
Detection System ◽  
Simultaneous Detection ◽  
Virus Detection ◽  
Control Measures ◽  
Analytical Sensitivity ◽  
Detection And Identification ◽  
One Step ◽  
Development And Validation

Tobamovirus species represent a threat to solanaceous crops worldwide, due to their extreme stability and being seed-borne. In particular, recent outbreaks of tomato brown rugose fruit virus in tomato and pepper crops led to the establishment of prompt control measures, and the need for reliable diagnosis was urged. Another member of the genus, tomato mottle mosaic virus, has recently risen attention due to reports in different continents and its common features with tomato brown rugose fruit virus. In this study, a new real-time RT-PCR detection system was developed for tomato brown rugose fruit virus and tomato mottle mosaic virus on tomato leaves and seeds using TaqMan chemistry. This test was designed to detect tomato mottle mosaic virus by amplifying the movement protein gene in a duplex assay with tomato brown rugose fruit virus target on the CP-3’NTR region, which was already validated as a single assay. The performance of this tests was evaluated, displaying analytical sensitivity 10-5-10-6-fold dilution for seeds and leaves, respectively, and good analytical specificity, repeatability, and reproducibility. Using the newly developed and validated test, tomato brown rugose fruit virus detection was 100% concordant with previously performed analyses on 106 official samples collected in 2021 from different continents.

A Real Time Control Architecture for Continuously Managing Patients in a Care Unit

1995 ◽  
Vol 34 (05) ◽  
pp. 475-488
Author(s):  
B. Seroussi ◽  
J. F. Boisvieux ◽  
V. Morice
Keyword(s):  
Real Time ◽  
Linear Time ◽  
Treatment Plan ◽  
Control Architecture ◽  
Real Time Control ◽  
Time Representation ◽  
Time Control ◽  
Continuous Support ◽  
Definition Of ◽  
Computerized System

Abstract:The monitoring and treatment of patients in a care unit is a complex task in which even the most experienced clinicians can make errors. A hemato-oncology department in which patients undergo chemotherapy asked for a computerized system able to provide intelligent and continuous support in this task. One issue in building such a system is the definition of a control architecture able to manage, in real time, a treatment plan containing prescriptions and protocols in which temporal constraints are expressed in various ways, that is, which supervises the treatment, including controlling the timely execution of prescriptions and suggesting modifications to the plan according to the patient’s evolving condition. The system to solve these issues, called SEPIA, has to manage the dynamic, processes involved in patient care. Its role is to generate, in real time, commands for the patient’s care (execution of tests, administration of drugs) from a plan, and to monitor the patient’s state so that it may propose actions updating the plan. The necessity of an explicit time representation is shown. We propose using a linear time structure towards the past, with precise and absolute dates, open towards the future, and with imprecise and relative dates. Temporal relative scales are introduced to facilitate knowledge representation and access.

In vitro and in vivo Assessment of Silver Nanoparticles Against Clostridium botulinum Type A Botulinum

2019 ◽  
Vol 16 (1) ◽  
pp. 113-119 ◽  
Author(s):  
Mohammad Aminianfar ◽  
Siavash Parvardeh ◽  
Mohsen Soleimani
Keyword(s):  
Silver Nanoparticles ◽  
Botulinum Toxin ◽  
Clostridium Botulinum ◽  
Lethal Dose ◽  
Type A ◽  
Positive Control ◽  
Negative Control ◽  
Laboratory Animals ◽  
Control Group ◽  
Nano Silver

Background: Clostridium botulinum causes botulism, a serious paralytic illness that results from the ingestion of a botulinum toxin. Because silver nanoparticle products exhibit strong antimicrobial activity, applications for silver nanoparticles in healthcare have expanded. Therefore, the objective of the current study was to assess a therapeutic strategy for the treatment of botulism toxicity using silver nanoparticles. Methods: A preliminary test was conducted using doses that produce illness in laboratory animals to determine the absolute lethal dose (LD100) of botulinum toxin type A (BoNT/A) in mice. Next, the test animals were divided into six groups containing six mice each. Groups I, II and III were the negative control (botulinum toxin only), positive control-1 (nano-silver only) and positive control-2 (no treatment), respectively. The remaining groups were allocated to the toxin that was supplemented with three nano-silver treatments. Results: The mortality rates of mice caused by BoNT/A significantly reduced in the treatment groups with different doses and injection intervals of nano-silver when compared to the negative control group. BoNT/A toxicity induced by intraperitoneal injection of the toxin of Clostridium botulinum causes rapid death while when coupled with nano-osilver results in delayed death in mice. Conclusion: These results, while open to future improvement, represent a preliminary step towards the satisfactory control of BoNT/A with the use of silver nanoparticles for human protection against this bioterrorism threat. Further study in this area can elucidate the underlying mechanism for detoxifying BoNT/A by silver nanoparticles.

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