Development of Rapid Extraction Method of Mycobacterium avium Subspecies paratuberculosis DNA from Bovine Stool Samples

Abstract

The rapid identification of Mycobacterium avium subspecies paratuberculosis (MAP) infected animals within the herd is essential for preventing the spread of the disease as well as avoiding human exposure. Although culture is seen as the gold standard, there are various molecular assays available i.e., polymerase chain reaction (PCR) or isothermal amplification technique (recombinase polymerase amplification (RPA)) for the detection of MAP. The accuracy of the molecular assays is highly dependent on the DNA extraction method. In order to establish a rapid point of need system for the detection of MAP DNA from stool samples, we developed a rapid DNA extraction protocol (MAP DNA SpeedXtract) specified for use in combination with the RPA. The whole procedure from “sample in” to “result out” was conducted in a mobile suitcase laboratory. The DNA extraction is based on reverse purification by magnetic beads, which reduces the required technical demand. The MAP DNA SpeedXtract was performed within 25 min and only three pipetting steps were needed. The amplification and detection time were 20 min in RPA. The sensitivity and specificity of the developed protocol in comparison with the lab-based silica membrane column extraction and real-time PCR were 90.9% (n = 22) and 100% (n = 23), respectively. In conclusion, we established a rapid and reliable protocol for the extraction and detection of MAP DNA. All reagents are cold chain independent. The entire setup is ideal for point of need identification of MAP infected cases.

Ultra-Low-Cost Integrated Silicon-based Transducer for On-Site, Genetic Detection of Pathogens

“…We will also add capabilities to communicate with smartphones to have access to the cloud so that the result of each diagnostic test can be passed to health agencies remotely. 37,38 To enable on-site testing, we will implement on-chip lysis capabilities (such as mechanical 39 or heatinduced cell lysis 40 ) to analyze the samples taken directly from the subject without further sample prep. We will also implement on-chip reverse transcription to enable analysis of RNA.…”

Multiplex Recombinase Polymerase Amplification Assay for Simultaneous Detection of Treponema pallidum and Haemophilus ducreyi in Yaws-Like Lesions

“…All critical RPA reagents thus enzymes/proteins and deoxy-nucleoside triphosphates are lyophilized into a single pellet which ensures the stability of reagents at room temperature for at least six months, reduces contamination, facilitates easy transportation of reagents and simplifies RPA applicability in low resource settings [ 23 , 24 ]. In addition, RPA assays have been evaluated with field friendly extraction techniques such as simple boiling of samples [ 24 , 25 , 26 ], SpeedXtract (Qiagen, Germany) [ 18 , 27 , 28 ] and GenoLyse DNA extraction Kit (Hain Life science, Nehren, Germany) [ 21 ] with little or no effect on RPA diagnostic performance. These DNA extraction techniques are less labor intensive with only one/two manipulation or pipetting steps and do not require sophisticated laboratory settings and hence applicable in low resource settings or mobile suitcase laboratories [ 18 , 27 , 28 ].…”

Molecular and Serological Footprints of Mycobacterium avium Subspecies Infections in Zoo Animals

“…Considering that MAP form clusters in different sample matrices [ 27 ], DNA was extracted from several portions of each pooled sample per animal species and environmental boot swab to compensate for nonhomogeneous bacterial distribution patterns. For RPA, 5 portions (~100 µg) of each fecal sample and 3 portions (~1 × 1 cm) of each boot swab were extracted using SpeedXtract (QIAgen, Hilden, Germany) and RPA, as previously described [ 28 , 29 ]. Thereafter, fecal and environmental samples were stored at −20 °C until shipped for laboratory screening with three real-time PCR assays targeting pan- Mycobacterium , MAP, MAA, and MAH.…”