High-Throughput Direct Fecal PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Sheep and Cattle

Plain, Karren M.; Marsh, Ian; Waldron, Anna M.; Galea, Francesca; Whittington, Ann-Michele; Saunders, Vanessa F.; Begg, Douglas J.; Silva, Kumudika de; Purdie, Auriol C.; Whittington, Richard J.

doi:10.1128/jcm.03233-13

Cited by 83 publications

(123 citation statements)

References 39 publications

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“…Hydrolysis probe quantitative PCR (qPCR) assays employ sequence-specific, fluorescently labeled probes attached to duplex-stabilizing molecules and are frequently used to accurately identify and quantify genetic sequences within clinical samples (29)(30)(31)(32). These assays can be multiplexed by using multiple sequencespecific probes attached to fluorescent molecules with unique and distinguishable emission spectra (33).…”

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confidence: 99%

Development and Validation of a Quantitative PCR Assay Using Multiplexed Hydrolysis Probes for Detection and Quantification of Theileria orientalis Isolates and Differentiation of Clinically Relevant Subtypes

et al. 2015

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T heileria orientalis is a vector-borne hemoprotozoan that infects cattle and buffalo and is generally spread by ticks of the Haemaphysalis genus (1-3). Historically, this organism has been referred to as Theileria sergenti, Theileria buffeli, or the T. orientalis/T. sergenti/T. buffeli complex; however, the name T. sergenti is now considered invalid (4), and T. orientalis is commonly used to refer to all (5). From a clinical perspective, T. orientalis can cause anemia, lethargy, jaundice, fever, abortion, and mortality in cattle (6). Clinical infection can also result in decreased milk production in dairy cattle (7). T. orientalis is a conditional pathogen, and while pathogenic forms are largely limited to Eastern Asia and Australasia (5, 6, 8-10), it is frequently detected in asymptomatic animals; these benign forms are globally spread (5,(11)(12)(13)(14). Analysis of the most common genotyping locus (p32), encoding the variable major piroplasm surface protein (MPSP), currently identifies 11 distinct T. orientalis genotypes (13,15,16). Of these genotypes, type 2 (Ikeda) and to a lesser extent type 1 (Chitose) are typically found in association with clinical disease (6,9,10,(17)(18)(19)(20)(21)(22). The presence of pathogenic and benign forms of T. orientalis greatly complicates its clinical diagnosis, with standard blood film analysis unable to identify the pathogenic genotypes.Multiple conventional PCR (cPCR) assays have been published for the identification of T. orientalis in blood samples (9,21,23,24). The most commonly cited assays detect and genotype T. orientalis by amplifying unique regions of the MPSP gene (21). These assays use two universal primers to detect T. orientalis infection and specific forward primers to identify the Ikeda, Chitose, and Buffeli genotypes. While this method is highly sensitive and has been validated in prior studies (19,21), it is not multiplexed and is highly susceptible to PCR inhibitors (6), which are often found in nucleotide extractions obtained from blood (25,26). To overcome inhibition, undiluted and diluted nucleotide extracts can be examined in parallel to prevent false-negative results (6,19,27). However, if multiple reactions per sample are required to determine the presence of infection, the procedure becomes both expensive and time-consuming. Furthermore, cPCR does not give an accurate representation of parasite load and therefore cannot provide an indication of the severity of T. orientalis infection. A recently developed multiplexed tandem PCR (28) is able to discriminate between four genotypes of T. orientalis, but it is only semiquantitative and therefore cannot be used to define a clinical threshold.Hydrolysis probe quantitative PCR (qPCR) assays employ sequence-specific, fluorescently labeled probes attached to duplex-

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confidence: 99%

Development and Validation of a Quantitative PCR Assay Using Multiplexed Hydrolysis Probes for Detection and Quantification of Theileria orientalis Isolates and Differentiation of Clinically Relevant Subtypes

et al. 2015

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“…The study found that replicate qPCR assays had variable detection rates when using samples that contained low MAP numbers. 7 The study also showed that the agreement between the HT-J PCR and radiometric culture decreased with decreasing numbers of MAP in the original sample, as culture was performed on a separate aliquot to what was used for HT-J PCR. 7 As such, the HT-J PCR has been recommended as a flock and herd test, rather than individual animal testing.…”

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confidence: 92%

“…7 The study also showed that the agreement between the HT-J PCR and radiometric culture decreased with decreasing numbers of MAP in the original sample, as culture was performed on a separate aliquot to what was used for HT-J PCR. 7 As such, the HT-J PCR has been recommended as a flock and herd test, rather than individual animal testing. 7 It is possible that the low numbers of FC-positive cows identified in our study was caused by the presence of subclinical infection in the study herd, therefore the fecal samples tested contained very low numbers of MAP, reducing the identification of infected animals.…”

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confidence: 92%

“…5 A 2014 study that investigated the HT-J PCR using Australian cattle and sheep populations found that in 870 cattle, 111 were FC positive and 124 were HT-J PCR positive, while in 507 sheep, 111 were FC positive and 117 were HT-J PCR positive. 7 However, that study 7 also attempted to identify fecal samples that were known to contain very low numbers of MAP, as the identification of subclinically infected animals is of particular importance with regard to maintenance of infection. The study found that replicate qPCR assays had variable detection rates when using samples that contained low MAP numbers.…”

mentioning

confidence: 99%

“…7 As such, the HT-J PCR has been recommended as a flock and herd test, rather than individual animal testing. 7 It is possible that the low numbers of FC-positive cows identified in our study was caused by the presence of subclinical infection in the study herd, therefore the fecal samples tested contained very low numbers of MAP, reducing the identification of infected animals. Using the manufacturer's recommended S/P ratio cutoff threshold of 0.15 and 1:20 dilution, the relative Se and Sp for colostrum and serum were not significantly different (i.e., relative Se was low and relative Sp was high for both samples).…”

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The diagnostic performance of an antibody enzyme-linked immunosorbent assay using serum and colostrum to determine the disease status of a Jersey dairy herd infected with Mycobacterium avium subspecies paratuberculosis

2015

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Colostrum may have the ability to improve the diagnostic accuracy of some tests when compared to serum for important livestock diseases because of the high concentrations of immunoglobulins present within this sample type. The ELISA for Johne’s disease is one such test, as it suffers from low sensitivity when testing serum samples collected during the subclinical stage of infection. Blood and colostrum samples were collected from 34 Jersey dairy cows and tested for antibodies against Mycobacterium avium subspecies paratuberculosis (MAP) by ELISA. Fecal samples were also collected and tested by a high-throughput Johne’s polymerase chain reaction (HT-J PCR) assay and fecal culture (FC), with the latter being used as the reference test. A receiver operating characteristic (ROC) analysis was performed, and the area under the curve (AUC) was calculated. The HT-J PCR and FC results were also compared. Of the 34 cows in this study, 4 had FC results consistent with MAP infection. The HT-J PCR did not identify any FC-positive cows. Using a 1:20 dilution and sample-to-positive (S/P) ratio cutoff threshold of 0.15, the relative sensitivity values of both serum (AUC 0. 56) and colostrum (AUC 0.63) were 0%. With lower sample dilutions, the relative sensitivity values of serum were 0% (1:2, AUC 0.62; 1:5, AUC 0.55); however, the relative sensitivity value of colostrum was 75% (95% confidence interval [CI]: 19–99%) at a dilution of 1:5, S/P ratio cutoff threshold of 0.15, and AUC of 0.73 (95% CI: 0.55–0.87). The testing of colostrum samples for MAP-specific antibodies by ELISA may provide improved identification of animals in the early stages of infection with MAP when compared with serum samples.

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Evaluation of an automated protocol for efficient and reliable DNA extraction of dietary samples

Wallinger

Staudacher

Sint

et al. 2017

Ecology and Evolution

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Molecular techniques have become an important tool to empirically assess feeding interactions. The increased usage of next‐generation sequencing approaches has stressed the need of fast DNA extraction that does not compromise DNA quality. Dietary samples here pose a particular challenge, as these demand high‐quality DNA extraction procedures for obtaining the minute quantities of short‐fragmented food DNA. Automatic high‐throughput procedures significantly decrease time and costs and allow for standardization of extracting total DNA. However, these approaches have not yet been evaluated for dietary samples. We tested the efficiency of an automatic DNA extraction platform and a traditional CTAB protocol, employing a variety of dietary samples including invertebrate whole‐body extracts as well as invertebrate and vertebrate gut content samples and feces. Extraction efficacy was quantified using the proportions of successful PCR amplifications of both total and prey DNA, and cost was estimated in terms of time and material expense. For extraction of total DNA, the automated platform performed better for both invertebrate and vertebrate samples. This was also true for prey detection in vertebrate samples. For the dietary analysis in invertebrates, there is still room for improvement when using the high‐throughput system for optimal DNA yields. Overall, the automated DNA extraction system turned out as a promising alternative to labor‐intensive, low‐throughput manual extraction methods such as CTAB. It is opening up the opportunity for an extensive use of this cost‐efficient and innovative methodology at low contamination risk also in trophic ecology.

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High-Throughput Direct Fecal PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Sheep and Cattle

Cited by 83 publications

References 39 publications

Development and Validation of a Quantitative PCR Assay Using Multiplexed Hydrolysis Probes for Detection and Quantification of Theileria orientalis Isolates and Differentiation of Clinically Relevant Subtypes

Development and Validation of a Quantitative PCR Assay Using Multiplexed Hydrolysis Probes for Detection and Quantification of Theileria orientalis Isolates and Differentiation of Clinically Relevant Subtypes

The diagnostic performance of an antibody enzyme-linked immunosorbent assay using serum and colostrum to determine the disease status of a Jersey dairy herd infected with Mycobacterium avium subspecies paratuberculosis

Evaluation of an automated protocol for efficient and reliable DNA extraction of dietary samples

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