Desenvolvimento e validação de uma reação em cadeia da polimerase quantitativa (qPCR) para diagnóstico da zoonose pseudovaríola bovina

Abstract

The pseudocowpox is a zoonosis caused by the pseudocowpox vírus (PCPV), classified within the genus Poxvirinae, Poxviridae family. The pseudocowpox affects mainly cows, also reaching suckling calves. In humans, it is a predominantly occupational disease, also known as "milkers `s nodule ", with occurrence of lesions mainly in fingers and hands of workers who deal directly with milking of infected animals. The lesions begin with appearance of redness spots, formation of papules, with possibility of ulceration and formation of crusts in infected epithelium. They are usually self-limited resolving within a few weeks but may become persistent in immunocompromised individuals. PCPV has been detected in several outbreaks throughout the country. In addition to public health damage, an outbreak of the disease in herds can lead to large economic losses, with possible disposal of milk and meat of infected animals. The diagnosis can be performed by electron microscopy, serological methods and by molecular biology techniques. Due to its sanitary and economic relevance, the official diagnosis of pseudocowpox in Brazil is carried out by the Ministry of Agriculture, Livestock and Food Supply (MAPA). This diagnosis is also part of the screening of suspicious samples for foot-andmouth disease, which constitutes a sanitary barrier of great economic importance forBrazil. Currently, detection of PCPV is performed by conventional PCR methods. This study aims to develop and validate a technique for the diagnosis of pseudocowpox using quantitative PCR (qPCR), which provides rapid responses regarding detection of the agent in suspect samples, with high sensitivity, specificity and reproducibility, among other validation requirements. The method developed was tested with known negative samples for the specificity assessment and runned against a panel of positive samples for sensitivity assessment. The limit of detection was evaluated from successive dilutions of the isolated reference virus and the standard plasmid containing the target DNA sequence. The repeatability of the test was also evaluated for the same analyst under the same conditions, as well as the reproducibility comparing the variance between different analysts or components of the qPCR. The method was able to detect up to 1000 copies / μL of PCPV in samples and had a specificity of 100% as well as a sensitivity of about 80%. The repeatability and reproducibility of the test were adequate according to the statistical parameters pre-established in official validation manuals. Therefore, the development of qPCR was satisfactory, showing itself adequate to the use in the diagnostic routine according to the national and international regulations related to the quality assurance and validation of a test method.