Comparison of nine DNA extraction methods for the diagnosis of bovine tuberculosis by real time PCR

Moura, A.R.; Hodon, Mikael Arrais; Filho, Paulo Martins Soares; Issa, Marina de Azevedo; Oliveira, Ana Paula Ferreira de; Júnior, Antônio Augusto Fonseca

doi:10.1590/0103-8478cr20151489

Cited by 9 publications

(10 citation statements)

References 15 publications

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“…Samples of the extracted DNA were submitted to quantitative PCR (qPCR) in the thermal cycler QuantStudio 7 Flex™ Real-Time PCR System 4 (Life Technologies, USA). The amplification reaction was performed according to Moura et al [19].…”

Section: Methodsmentioning

confidence: 99%

Intercurrence of Paratuberculosis in Intradermal Tuberculin Test Reactive Cattle

Souza

Soares

Medeiros-Ronchi

et al. 2020

Acta Scientiae. Vet.

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Background: Bovine tuberculosis control programs are based on a standard diagnostic method, the intradermal test with purified protein derivatives, which is used to identify and eliminate diseased animals. Currently none of the tests available allow complete differentiation between infected and uninfected animals. The main limitations of the tests available are related to diagnostic sensitivity and specificity, which results in false positive reactions due to the existence of cross infections, and also false negative, inherent to the state of energy of some animals. The aim of this work was to study the intercurrence of paratuberculosis in tuberculosis reactive cattle by the comparative cervical test.Materials, Methods & Results: Three hundred and thirty four cattle were evaluated using the comparative cervical test (CCT) and serology for tuberculosis (TB) and paratuberculosis (PTB) ELISA IDEXX®. All of the animals testing positive by CCT were euthanized and necropsied. Fragments of lymph node, lung and intestine were collected and analyzed using histopathological techniques, with staining by Hematoxylin-Eosin (HE). Samples of lung and lymph nodes (retropharyngeal, submandibular, cervical and mediastinal) of the animals testing positive by CCT were evaluated using qPRC for M. bovis, and intestinal and mesenteric lymph nodes using PCR for PTB. Of the 334 cattle evaluated using the comparative cervical test, 16 were considered positive. No lesions suggestive of tuberculosis were found in the macroscopic inspection of the carcasses. The most evident anatomical and pathological finding was a thickening of intestinal mucosa, found in 12 of the 16 cattle submitted to necropsy. No microscopic lesions suggestive of TB were identified nor was the presence of M. bovis detected by qPCR. The main histopathological findings were observed in the small intestine and mesenteric lymph nodes and identified as enteritis, lymphangitis, lymphangiectasia and granulomatous lymphadenitis. In the intestine the changes are characterized by dilated and inflamed lymphatic vessels and intense inflammatory infiltrate on the mucosa and submucosa. Of the 334 serum samples evaluated, the M. bovis ELISA Antibody Test (IDEXX®) identified 17 positive animals. All the cattle considered positive by M. bovis ELISA were considered negative by CCT. In the samples from nine animals (9/16), DNA from M. avium subsp. paratuberculosis (MAP) was identified and in twelve carcasses (12/16) lesions characteristic of PTB were found, which were subsequently confirmed by histopathological techniques. In another nine animals of the herd anti-MAP antibodies were detected. None of those that tested positive by PTB ELISA were reactive by CCT.Discussion: Animals considered positive by TB ELISA that were not positive in the intradermal test does not mischaracterize the clinical picture of the disease. Considering the inverse relationship between cell-mediated and humoral responses to M. bovis, the intradermal test and the serological tests are designed to measure different immunological responses, which develop during different stages of infection. The progress of the cellular immunological response to humoral immunity occurs in the most advanced stages of tuberculosis. Of the 16 cattle considered positive by CCT, 12 animals presented macroscopic and histological lesions suggestive of PTB and DNA from MAP was detected in nine. Although it is the official test for the control of TB in different countries, the intradermal test with PPD has presented limitations, primarily related to specificity. M. avium subsp. Paratuberculosis is considered the main cause of false positive reactions in the intradermal test. The PPD bacterial extract is a complex mixture of proteins, lipids, sugars and nucleic acids, and many of these components are also shared by numerous species of mycobacteria (tuberculous or not).

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Section: Methodsmentioning

confidence: 99%

Intercurrence of Paratuberculosis in Intradermal Tuberculin Test Reactive Cattle

Souza

Soares

Medeiros-Ronchi

et al. 2020

Acta Scientiae. Vet.

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“…Oligonucleotide primers and probes were procured from IDT (Coralville, IA, USA). Real time PCR has previously been described as an effective method by which to assess the performance of extraction methods in that it is highly specific, it can detect very low levels of target and can indirectly inform on the purity of extracted samples [17][18][19]. The core amplification reagent used in all real time RT-PCR assays was qScript XLT 1-Step RT-qPCR ToughMix (Quanta Bio, Gaithersburg, MD, USA).…”

Section: Reference Methods For Rt-pcr Detection Of Nucleic Acid Targementioning

confidence: 99%

“…Multiple studies on the performance of NA platforms have been described previously thus this work utilized real time PCR analyses of sample extracts to establish if sufficient amounts and purity of target NAs were generated to give a positive result [17][18][19]. Human raw sputum, whole blood and stool specimen matrices were used in this study because they are common specimen types used for the diagnosis of many infectious diseases and because each is considered complex because of physiochemical attributes including viscosity, heterogeneity, extraneous NAs, commensal microflora, cellular debris and the presence of PCR inhibitors [20][21][22][23][24][25][26].…”

Section: Specimen Matricesmentioning

confidence: 99%

Performance and workflow assessment of six nucleic acid extraction technologies for use in resource limited settings

et al. 2019

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Infectious disease nucleic acid amplification technologies (NAAT) have superior sensitivity, specificity, and rapid time to result compared to traditional microbiological methods. Recovery of concentrated, high quality pathogen nucleic acid (NA) from complex specimen matrices is required for optimal performance of several NA amplification/detection technologies such as polymerase chain reaction (PCR). Fully integrated NAAT platforms that enable rapid sample-to-result workflows with minimal user input are generally restricted to larger reference lab settings, and their complexity and cost are prohibitive to widespread implementation in resource limited settings (RLS). Identification of component technologies for incorporation of reliable and affordable sample preparation with pathogen NA amplification/detection into an integrated platform suitable for RLS, is a necessary first step toward achieving the overarching goal of reducing infectious disease-associated morbidity and mortality globally. In the current study, we evaluate the performance of six novel NA extraction technologies from different developers using blinded panels of stool, sputum and blood spiked with variable amounts of quality-controlled DNA- and/or RNA-based microbes. The extraction efficiencies were semi-quantitatively assessed using validated real-time reverse transcription (RT)-PCR assays specific for each microbe and comparing target-specific RT-PCR results to those obtained with reference NA extraction methods. The technologies were ranked based on overall diagnostic accuracy (analytical sensitivity and specificity). Sample input and output volumes, total processing time, user-required manual steps and cost estimates were also examined for suitability in RLS. Together with the performance analysis, these metrics were used to select the more suitable candidate technologies for further optimization of integrated NA amplification and detection technologies for RLS.

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“…Despite the cards showed partial lysis for some gram-positive species and Mycobacterium species, inactivation was observed for cell suspensions with densities below ≤ 10 4 cfu ml − 1 [6]. Furthermore, the cards presented good e cacy to store and to transport samples for the direct detection of Mycobacterium tuberculosis from sputum specimens [7], and to extract DNA of M. bovis from bovine tissues [8].…”

Section: Introductionmentioning

confidence: 99%

Assessment of gross lesions preservation methods for the direct diagnosis of bovine tuberculosis: a constant search for improvement

Cárdenas

Ikuta

Rabaquim

et al. 2021

Preprint

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Objectives The present study assessed the sensitivity and biosafety of the FTA Elute Card® (FTA) (GE Healthcare) as an alternative preservation method of gross lesions for bovine tuberculosis diagnosis, that can endure large distances and high environmental temperatures, which are found in the Brazilian territory. Results The FTA card was compared to the freezing and sodium borate solution methods to preserve 134 gross lesions samples from slaughterhouses located in three Brazilian states. The calculated value for the sensitivity of sodium borate solution (SBS) stored for 30 days was higher than those estimated for the methods of freezing and SBS stored for 60 days, when the PCR and isolation results were interpreted in parallel (0.55). The FTA cards presented the lowest diagnostic sensitivity and did not inactivate all mycobacteria. Given the rush of a slaughterhouse routine, the quantity of lesions with different pathological stages cannot be measured on each FTA card. Thus, as a preservation method of gross tuberculosis lesions, the FTA cards can present infection risks for people who have direct or indirect contact with them.

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Comparison of nine DNA extraction methods for the diagnosis of bovine tuberculosis by real time PCR

Abstract: Bovine tuberculosis is an infectious disease with a high impact on the cattle industry, particularly in developing countries. PCR is a very sensitive method for detection of infectious agents

Cited by 9 publications

References 15 publications

Intercurrence of Paratuberculosis in Intradermal Tuberculin Test Reactive Cattle

Intercurrence of Paratuberculosis in Intradermal Tuberculin Test Reactive Cattle

Performance and workflow assessment of six nucleic acid extraction technologies for use in resource limited settings

Assessment of gross lesions preservation methods for the direct diagnosis of bovine tuberculosis: a constant search for improvement

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