ABSTRACT
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.
Subject(s)
Chagas Disease , Trypanosoma cruzi , Trypanosoma cruzi/genetics , Trypanosoma cruzi/isolation & purification , Chagas Disease/epidemiology , Chagas Disease/parasitology , Chagas Disease/transmission , Chagas Disease/diagnosis , Animals , Real-Time Polymerase Chain Reaction/methods , Euterpe , Brazil/epidemiology , Humans , DNA, Protozoan/geneticsABSTRACT
Three commercial kits of One-Step RT-qPCR were evaluated for the molecular diagnosis of Canine Distemper Virus. Using the kit that showed better performance, two systems of Real-time RT-PCR (RT-qPCR) assays were tested and compared for analytical sensitivity to Canine Distemper Virus RNA detection: a One-Step RT-qPCR (system A) and a One-Step RT-qPCR combined with NESTED-qPCR (system B). Limits of detection for both systems were determined using a serial dilution of Canine Distemper Virus synthetic RNA or a positive urine sample. In addition, the same urine sample was tested using samples with prior centrifugation or ultracentrifugation. Commercial kits of One-Step RT-qPCR assays detected canine distemper virus RNA in 10 (100%) urine samples from symptomatic animals tested. The One-Step RT-qPCR kit that showed better results was used to evaluate the analytical sensitivity of the A and B systems. Limit of detection using synthetic RNA for the system A was 11 RNA copies µL-1 and 110 RNA copies µl-1 for first round System B. The second round of the NESTED-qPCR for System B had a limit of detection of 11 copies µl-1. Relationship between Ct values and RNA concentration was linear. The RNA extracted from the urine dilutions was detected in dilutions of 10-3 and10-2 by System A and B respectively. Urine centrifugation increased the analytical sensitivity of the test and proved to be useful for routine diagnostics. The One-Step RT-qPCR is a fast, sensitive and specic method for canine distemper routine diagnosis and research projects that require sensitive and quantitative methodology.(AU)
Três kits comerciais de One-Step RT-qPCR foram avaliados para o diagnóstico molecular do Vírus da Cinomose Canina.Utilizando o kit que apresentou melhor desempenho, dois sistemas de RT-PCR em tempo real (RT-qPCR) foram comparados quanto à sensibilidade analítica na detecção do RNA do Vírus da Cinomose Canina:One-Step RT-qPCR (Sistema A) e One-Step RT-qPCR seguido da NESTED-qPCR (Sistema B).Os limites de detecção dos dois sistemas foram determinados utilizando diluição seriada de RNA sintético do Vírus da Cinomose Canina ou de uma amostra de urina positiva. Adicionalmente, uma amostra de urina foi avaliada com centrifugação ou ultracentrifugação prévia. Os kits comerciais de One-Step RT-qPCR amplificaram o RNA do vírus da cinomose canina em 10 (100%) amostras de urinas de animais sintomáticos. O kit de One-Step RT-qPCR que apresentou melhor resultado foi utilizado para avaliar a sensibilidade analítica dos sistemas A e B. Na reação da curva padrão com RNA sintético, o limite de detecção do sistema A foi de 11 cópias de RNA µL-1. No sistema B foi de 110 cópias de RNA µL-1 na One-Step RT-qPCR e 11 cópias de RNA µL-1 na NESTED-qPCR. A relação entre os valores de Ct e concentração de RNA foi linear. O RNA extraído das diluições da urina foi detectado nas diluições de 10-3 e10-2 pelos sistemas A e B, respectivamente. A centrifugação prévia da urina aumentou a sensibilidade analítica da análise e mostrou ser importante para a rotina diagnóstica. A reação de One-Step RT-PCR é um método rápido, sensível, específico e aplicável na rotina de diagnóstico molecular da cinomose e em projeto de pesquisa que requer metodologia quantitativa e sensível.(AU)
Subject(s)
Animals , Dogs , Dog Diseases , Distemper Virus, Canine , Distemper/diagnosis , Diagnostic Techniques and ProceduresABSTRACT
ABSTRACT: Three commercial kits of One-Step RT-qPCR were evaluated for the molecular diagnosis of Canine Distemper Virus. Using the kit that showed better performance, two systems of Real-time RT-PCR (RT-qPCR) assays were tested and compared for analytical sensitivity to Canine Distemper Virus RNA detection: a One-Step RT-qPCR (system A) and a One-Step RT-qPCR combined with NESTED-qPCR (system B). Limits of detection for both systems were determined using a serial dilution of Canine Distemper Virus synthetic RNA or a positive urine sample. In addition, the same urine sample was tested using samples with prior centrifugation or ultracentrifugation. Commercial kits of One-Step RT-qPCR assays detected canine distemper virus RNA in 10 (100%) urine samples from symptomatic animals tested. The One-Step RT-qPCR kit that showed better results was used to evaluate the analytical sensitivity of the A and B systems. Limit of detection using synthetic RNA for the system A was 11 RNA copies µL-1 and 110 RNA copies µl-1 for first round System B. The second round of the NESTED-qPCR for System B had a limit of detection of 11 copies µl-1. Relationship between Ct values and RNA concentration was linear. The RNA extracted from the urine dilutions was detected in dilutions of 10-3 and10-2 by System A and B respectively. Urine centrifugation increased the analytical sensitivity of the test and proved to be useful for routine diagnostics. The One-Step RT-qPCR is a fast, sensitive and specific method for canine distemper routine diagnosis and research projects that require sensitive and quantitative methodology.
RESUMO: Três kits comerciais de One-Step RT-qPCR foram avaliados para o diagnóstico molecular do Vírus da Cinomose Canina.Utilizando o kit que apresentou melhor desempenho, dois sistemas de RT-PCR em tempo real (RT-qPCR) foram comparados quanto à sensibilidade analítica na detecção do RNA do Vírus da Cinomose Canina:One-Step RT-qPCR (Sistema A) e One-Step RT-qPCR seguido da NESTED-qPCR (Sistema B).Os limites de detecção dos dois sistemas foram determinados utilizando diluição seriada de RNA sintético do Vírus da Cinomose Canina ou de uma amostra de urina positiva. Adicionalmente, uma amostra de urina foi avaliada com centrifugação ou ultracentrifugação prévia. Os kits comerciais de One-Step RT-qPCR amplificaram o RNA do vírus da cinomose canina em 10 (100%) amostras de urinas de animais sintomáticos. O kit de One-Step RT-qPCR que apresentou melhor resultado foi utilizado para avaliar a sensibilidade analítica dos sistemas A e B. Na reação da curva padrão com RNA sintético, o limite de detecção do sistema A foi de 11 cópias de RNA µL-1. No sistema B foi de 110 cópias de RNA µL-1 na One-Step RT-qPCR e 11 cópias de RNA µL-1 na NESTED-qPCR. A relação entre os valores de Ct e concentração de RNA foi linear. O RNA extraído das diluições da urina foi detectado nas diluições de 10-3 e10-2 pelos sistemas A e B, respectivamente. A centrifugação prévia da urina aumentou a sensibilidade analítica da análise e mostrou ser importante para a rotina diagnóstica. A reação de One-Step RT-PCR é um método rápido, sensível, específico e aplicável na rotina de diagnóstico molecular da cinomose e em projeto de pesquisa que requer metodologia quantitativa e sensível.