SYBR Green qPCR Technique for the Detection of<i>Trypanosoma cruzi</i>in Açaí Pulp

Cardoso, Gabrielle Virgínia Ferreira; Lima, Joelson Sousa; Oliveira, Andrey Carlos do Sacramento de; Silva, Josyane Brasil da; Roos, Talita Bandeira; Moraes, Carina Martins de

doi:10.1089/fpd.2019.2745

Cited by 7 publications

(5 citation statements)

References 20 publications

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“…Namely, the characteristic dark color of the fruit, which is associated with high anthocyanin concentrations and a large amount of organic matter [34,35], greatly limit the possibility of parasites detection during microscopic visualization. In this context, molecular methods can overcome these limitations regarding analysis directly from food, and provide specific diagnosis [26,41,50,52,63] and T. cruzi genotyping [51]. The qPCR developed in the present study showed improved sensitivity, in which was possible to detect the DNA corresponding to 0.01 T. cruzi equivalents/mL in the sample.…”

Section: Discussionmentioning

confidence: 87%

“…Ferreira et al[51], in order to evaluate the amplifiability of DNA in a conventional PCR, employed a plant-specific primer pair[78], which encodes the ribulose 1,5-diphosphate carboxylase/oxygenase gene (rbcL) of the plant chloroplast. However, to date, published studies related to the development of a methodology based on Real-Time quantitative PCR, for the T. cruzi diagnosis in food samples, have not included an internal amplification control, despite using negative and positive controls in the reactions[26,50,52]. In our study, we used a synthetic DNA from a commercial kit (Applied Biosystems, Foster City, CA, USA) to monitor the efficiency of the DNA extraction and the absence of inhibitors at qPCR.…”

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confidence: 99%

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Validation of a novel multiplex real-time PCR assay forTrypanosoma cruzidetection and quantification in açai pulp

Finamore-Araujo

Faier-Pereira

Brito

et al. 2020

Preprint

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In Brazil, orally acquired T. cruzi infection has become the most relevant transmission mechanisms from public health perspective. Around 70% of new Chagas disease cases have been associated with consumption of contaminated food or beverages. Açai (Euterpe oleracea and Euterpe precatoria) is currently one of the most commercialized Amazonian fruits in the Brazilian and international markets. Therefore, it has become important to incorporate in the production process some procedures to measure out effective hygiene and product quality control required by global market. Molecular methods have been developed for rapid detection and quantification of T. cruzi DNA in several biological samples, including food matrices, for epidemiological investigation of Chagas disease and food quality control. However, a high-performance molecular methodology since DNA extraction until detection and quantification of T. cruzi DNA in açai berry pulp is still needed. Herein, a simple DNA extraction methodology was standardized from the supernatant of açai berry pulp stabilized in a Lysis buffer. In addition, a multiplex real time qPCR assay, targeting T. cruzi DNA and an Exogenous Internal Positive Control was developed and validated, using reference from all T. cruzi DTUs and commercial samples of açai pulp, from an endemic municipality with previous history of oral Chagas disease outbreak. Thus, a high-sensitivity qPCR assay, that could detect up to 0.01 parasite equivalents/mL in açai, was reached. As of the 45 commercial samples analyzed, 9 (20%) were positive for T. cruzi. This high-sensitive, fast and easy-to-use molecular assay is compatible with most of the laboratories involved in the investigations of oral Chagas disease outbreaks, representing an important tool to the epidemiology, control and surveillance of Chagas disease.Author SummaryOral transmission of Chagas disease has acquired an increasingly importance on the disease epidemiology. Most of the orally acquired Chagas Disease cases are related to the consumption of fresh foods or drinks, as sugar cane juice, açai berry pulp and bacaba wine, contaminated with triatomines or its feces. In Brazil, it has recently caused numerous outbreaks and has been linked to unusually severe acute infections. So far, the evaluation of the potential for oral transmission of Chagas disease through the consumption of açai-based products is mostly determined by clinical or parasitological methods. Despite the recent advances, a highly sensitive, reproductible and properly validated real time PCR assay for the molecular diagnostic of T. cruzi in açai pulp samples is still missing. Herein, a simple and reproducible multiplex real-time PCR assay was developed to the detection and quantification of T. cruzi DNA in açai pulp samples. This methodology, that includes a simple step for sample stabilization and DNA extraction based on silica-membrane spin columns, can be useful for analyzing orally transmitted acute Chagas disease outbreaks.

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Section: Discussionmentioning

confidence: 87%

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confidence: 99%

Validation of a novel multiplex real-time PCR assay forTrypanosoma cruzidetection and quantification in açai pulp

Finamore-Araujo

Faier-Pereira

Brito

et al. 2020

Preprint

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“…However, these methods are laborious, time-consuming, and more subject to contamination [33]. Some studies were effective in identifying T. cruzi in food samples [9,18,[20][21][22][23]34]. Recently, our group developed an easily, reproducible, fast, and sensitive multiplex qPCR method that is capable of identifying the parasite's DNA in açaí pulp samples [19].…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, the lack of hygiene during the harvesting and handling stage of the fruit is one of the main targets of T. cruzi contamination, leading açaí to be the food associated with the highest number of cases of oral outbreaks of CD in the Brazilian Amazon region in recent years [12,17]. The use of PCR for the detection of T. cruzi DNA from açai has already been described in the literature, specifically in studies showing that the molecular detection technique has greater sensitivity compared to other detection methods [9,[18][19][20][21][22][23]. However, a recurrent issue regarding the use of DNA for pathogen detection is the correlation with its viability [24,25].…”

Section: Introductionmentioning

confidence: 99%

The Development of a One-Step RT-qPCR for the Detection and Quantification of Viable Forms of Trypanosoma cruzi in Açai Samples from Areas at Risk of Chagas Disease through Oral Transmission

Faier-Pereira,

Finamore-Araujo,

Brito

et al. 2024

IJMS

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Currently, approximately 70% of new cases of Chagas disease (CD) in Brazil are attributed to oral transmission, particularly through foods such as açaí, bacaba, and sugarcane juice, primarily in the northern and northeastern regions of the country. This underscores the imperative need to control the spread of the disease. The methods utilized to conduct quality control for food associated with outbreaks and to assess the potential for the oral transmission of CD through consuming açaí primarily rely on isolating the parasite or inoculating food into experimental animals, restricting the analyses to major research centers. While there are existing studies in the literature on the detection and quantification of T. cruzi DNA in açaí, the evaluation of parasites’ viability using molecular methods in this type of sample and differentiating between live and dead parasites in açaí pulp remain challenging. Consequently, we developed a molecular methodology based on RT-qPCR for detecting and quantifying viable T. cruzi in açaí pulp samples. This protocol enables the stabilization and preservation of nucleic acids in açaí, along with incorporating an exogenous internal amplification control. The standardization of the RNA extraction method involved a simple and reproducible approach, coupled with a one-step RT-qPCR assay. The assay underwent validation with various T. cruzi DTUs and demonstrated sensitivity in detecting up to 0.1 viable parasite equivalents/mL in açaí samples. Furthermore, we investigated the effectiveness of a bleaching method in eliminating viable parasites in açaí samples contaminated with T. cruzi by comparing the detection of DNA versus RNA. Finally, we validated this methodology using açaí pulp samples positive for T. cruzi DNA, which were collected in a municipality with a history of oral CD outbreaks (Coari-AM). This validation involved comparing the detection and quantification of total versus viable T. cruzi. Collectively, our findings demonstrate the feasibility of this methodology in detecting viable forms of T. cruzi in açaí pulp samples, emerging as a crucial tool for monitoring oral outbreaks of Chagas disease resulting from açaí consumption.

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“…Conventional PCR can only detect one pathogen at a time, but there are many pathogenic bacteria in food ( Kim and Kim, 2021 ). Therefore, based on conventional PCR, dozens of different types of PCR methods were derived, mainly including multiple polymerase chain reaction (mPCR), real-time quantitative polymerase chain reaction (qPCR), and digital polymerase chain reaction technology (dPCR) ( Tang et al, 2018 ; Mou et al, 2019a ; Lei et al, 2020a ; Cardoso et al, 2020 ; Huang et al, 2021a ).…”

Section: Molecular Biology Detection Technologymentioning

confidence: 99%

Research progress on detection techniques for point-of-care testing of foodborne pathogens

Zhao

Huang

et al. 2022

Front. Bioeng. Biotechnol.

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The global burden of foodborne disease is enormous and foodborne pathogens are the leading cause of human illnesses. The detection of foodborne pathogenic bacteria has become a research hotspot in recent years. Rapid detection methods based on immunoassay, molecular biology, microfluidic chip, metabolism, biosensor, and mass spectrometry have developed rapidly and become the main methods for the detection of foodborne pathogens. This study reviewed a variety of rapid detection methods in recent years. The research advances are introduced based on the above technical methods for the rapid detection of foodborne pathogenic bacteria. The study also discusses the limitations of existing methods and their advantages and future development direction, to form an overall understanding of the detection methods, and for point-of-care testing (POCT) applications to accurately and rapidly diagnose and control diseases.

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SYBR Green qPCR Technique for the Detection ofTrypanosoma cruziin Açaí Pulp

Cited by 7 publications

References 20 publications

Validation of a novel multiplex real-time PCR assay forTrypanosoma cruzidetection and quantification in açai pulp

Validation of a novel multiplex real-time PCR assay forTrypanosoma cruzidetection and quantification in açai pulp

The Development of a One-Step RT-qPCR for the Detection and Quantification of Viable Forms of Trypanosoma cruzi in Açai Samples from Areas at Risk of Chagas Disease through Oral Transmission

Research progress on detection techniques for point-of-care testing of foodborne pathogens

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