New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis

BACKGROUND: Johne's disease, a serious chronic form of enteritis in ruminants, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). As the organism is very slow-growing and fastidious, several PCR-based methods for detection have been developed, based mainly on the MAP-specific gene IS900. However, because this gene is similar to genes in other mycobacteria, there is a need for sensitive and reliable methods to confirm the presence of MAP. As described here, two new real-time PCR systems on the IS900 gene and one on the F57 gene were developed and carefully validated on 267 strains and 56 positive clinical faecal samples. RESULTS: Our confirmatory PCR systems on IS900 were found sensitive and specific, only yielding weak false positive reactions in one strain for each system. The PCR system on F57 did not elicit any false positives and was only slightly less sensitive than our primary IS900-system. DNA from both naturally infected and spiked faeces that tested positive with our primary system could be confirmed with all new systems, except one low-level infected sample that tested negative with the F57 system. CONCLUSION: We recommend using the newly constructed DH3 PCR system on the F57 gene as the primary confirmatory test for PCR positives, but should it fail due to its lower sensitivity, the DH1 and DH2 PCR systems should be used

Rapid Identification of Bio-Molecules Applied for Detection of Biosecurity Agents Using Rolling Circle Amplification

Detection and identification of pathogens in environmental samples for biosecurity applications are challenging due to the strict requirements on specificity, sensitivity and time. We have developed a concept for quick, specific and sensitive pathogen identification in environmental samples. Target identification is realized by padlock- and proximity probing, and reacted probes are amplified by RCA (rolling-circle amplification). The individual RCA products are labeled by fluorescence and enumerated by an instrument, developed for sensitive and rapid digital analysis. The concept is demonstrated by identification of simili biowarfare agents for bacteria (Escherichia coli and Pantoea agglomerans) and spores (Bacillus atrophaeus) released in field

Detection and confirmation of Mycobacterium avium subsp. paratuberculosis in clinical samples

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of the serious disease paratuberculosis in ruminants. It is a chronic enteric disease that causes considerable economic losses worldwide. Paratuberculosis leads to reduced milk production and eventually, diarrhoea, weight loss and death. Its slow development, the inappropriate immune response of the host and the fastidiousness of the bacteria all contribute to the difficulty of early diagnosis, necessary to restrict spread of the disease. Thanks to rigorous control measures, paratuberculosis is rare or absent in Sweden. However, occasional import-related outbreaks have occurred, during which all animals in the infected herds were culled. Freedom from paratuberculosis is mainly monitored by slow culture methods. In some situations, fast and reliable alternative methods are needed, for instance, when semen is imported for breeding purposes. Donor bulls may be asymptomatic carriers of MAP and the risk for venereal transmission of the disease is insufficiently investigated. In this thesis, the development and sensitivity assessment of a protocol for detection of MAP in bovine semen by real-time PCR is described. Beadbeating with zirconia/silica beads and phenol/chloroform extraction was used to purify the bacterial DNA and it was shown to successfully remove PCR inhibiting substances. A method for accurately evaluating the analytical sensitivity was also developed. By analysis of artificially infected samples, a sensitivity of 10 organisms per 100 µl sample was assessed. The target gene, for the method described here and for many other PCR methods for detection of MAP, is the insertion element IS900. Although specific for MAP, it has been shown to share similarities with genes in other mycobacteria. Positive PCR results must, therefore, be confirmed by an alternative method. Three novel real-time PCR systems were designed and validated on 267 strains and 58 positive clinical faecal and tissue samples. Two systems were based on IS900 and one system on the MAP-specific gene F57. The latter was the most specific for MAP and is therefore the recommended system for confirmation, but as it was slightly less sensitive when tested on pure DNA, the other systems may be applied when necessary

Molecular diagnostic methods for Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of the chronic enteric disease paratuberculosis in ruminants that causes considerable economic losses worldwide. Due to rigorous control measures, paratuberculosis is rare or absent in Sweden. However, import-related outbreaks have occurred. Diagnostic surveillance and outbreak investigations are mainly carried out by very slow culture methods. Faster and equally reliable molecular methods are needed for detection of MAP in several clinical matrixes. MAP is primarily shed in faeces, the most important testing material. The abundance of PCR inhibitory substances in faeces constitutes a diagnostic challenge. Semen, imported for breeding purposes, may contain MAP if the donor bulls are asymptomatic carriers. MAP has been suggested as the causative agent of Crohn's disease and its presence in consumers' milk might be of concern. In this thesis, the development and sensitivity assessment of protocols for detection of MAP in ruminant faeces, semen and milk by real-time PCR are described. The analytical sensitivities were assessed to 104 MAP/g faeces, 10 MAP/100 µl semen and 100 MAP/ml milk. The faeces direct PCR was validated on 202 proficiency test samples. MAP was detected in 97% of previously frozen positive samples - better than culture. Pellet and cream fractions of milk were pooled before cell lysis and DNA extraction by automated magnetic bead separation. In a study of 56 dairy herds, tank milk PCR was compared to culture of environmental faecal samples for herd prevalence testing. By the latter, 68% of the herds were positive, while 30% were positive by PCR. Due to the concluded low abundance of MAP in milk tanks, milk PCR would be more useful for testing of MAP in consumers’ milk, than for herd prevalence testing. Three real-time PCR systems were designed for confirmation of PCR positives and validated on 267 strains and 58 positive faecal and tissue samples. The system based on the gene F57 was the most specific. A faecal culture screening of 501 wild guanacos in Chile yielded MAP colonies from 21 guanacos (4.2%), representing the first isolation of MAP from wild animals in the Chilean Patagonia. Confirmation was done by PCR and typing was performed by PCR-REA. All strains proved to be of C type

Detection of Rotavirus Using Padlock Probes and Rolling Circle Amplification

Rotavirus infections are one of the most common reasons for hospitalizations due to gastrointestinal diseases. Rotavirus is often diagnosed by latex agglutination assay, chromatography immunoassay, or by electron microscopy, which are all quite insensitive. Reverse transcription polymerase chain reaction, on the other hand, is very sensitive to variations at the genomic level. We developed a novel assay based on a set of 58 different padlock probes with a detection limit of 1,000 copies. Twenty-two patient samples were analyzed and the assay showed high concordance with a PCR-based assay. In summary, we present a new assay for sensitive and variation tolerant detection of rotavirus

Detection of Rifampicin Resistance in Mycobacterium tuberculosis by Padlock Probes and Magnetic Nanobead- Based Readout

Control of the global epidemic tuberculosis is severely hampered by the emergence of drug-resistant Mycobacterium tuberculosis strains. Molecular methods offer a more rapid means of characterizing resistant strains than phenotypic drug susceptibility testing. We have developed a molecular method for detection of rifampicin-resistant M. tuberculosis based on padlock probes and magnetic nanobeads. Padlockprobes were designed to target the most common mutations associated with rifampicinresistance in M. tuberculosis, i.e. at codons 516, 526 and 531 in the gene rpoB. Fordetection of the wild type sequence at all three codons simultaneously, a padlock probe and two gap-fill oligonucleotides were used in a novel assay configuration, requiring three ligation events for circularization. The assay also includes a probe for identificationof the M. tuberculosis complex. Circularized probes were amplified by rolling circle amplification. Amplification products were coupled to oligonucleotide-conjugatedmagnetic nanobeads and detected by measuring the frequency-dependent magneticresponse of the beads using a portable AC susceptometer

List of oligonucleotides used in this assay.

a<p>A + sign before nt indicate the LNA residues. As implicated in PLP_A: the bold font indicated the Rotavirus targeting padlock probe arms, Italic font indicates the AluI restriction site, and the underlined sequence the sequence for detection probe hybridization.</p><p>List of oligonucleotides used in this assay.</p

A General Method for Rapid Determination of Antibiotic Susceptibility and Species in Bacterial Infections

To ensure correct antibiotic treatment and reduce the unnecessary use of antibiotics, there is an urgent need for new rapid methods for species identification and determination of antibiotic susceptibility in infectious pathogenic bacteria. We have developed a general method for the rapid identification of the bacterial species causing an infection and the determination of their antibiotic susceptibility profiles. An initial short cultivation step in the absence and presence of different antibiotics was combined with sensitive species-specific padlock probe detection of the bacterial target DNA to allow a determination of growth (i.e., resistance) and no growth (i.e., susceptibility). A proof-of-concept was established for urinary tract infections in which we applied the method to determine the antibiotic susceptibility profiles of Escherichia coli for two drugs with 100% accuracy in 3.5 h. The short assay time from sample to readout enables fast appropriate treatment with effective drugs and minimizes the need to prescribe broad-spectrum antibiotics due to unknown resistance profiles of the treated infection.De två sista författarna delar sistaförfattarskapet.</p