Sagarika Kanjilal

Performance evaluation of five commercial real-time PCR reagent systems using TaqMan assays for B. anthracis detection

BACKGROUND Real-time PCR assay sensitivity is affected by the choice and concentrations of reaction mix constituents among other factors such as primers, probes, and analytical assay platforms. Commercially available reagent mixes facilitate PCR assay set-up with fewer steps and timeliness. However, determination of analytical assay framework is important for ready-to-use real-time PCR reagent systems for rapid, quantitative and accurate detection of bioterror pathogens such as Bacillus anthracis. METHODS In this study, performance characteristics of five commercially available quantitative PCR reagent mixes were evaluated using TaqMan-based real-time PCR. The reagent systems were tested for compatibility on the ABI 7000 assay platform and compared for their distinctive analytical characteristics using the B. anthracis rpoB and pag gene real-time PCR assays. RESULTS AND CONCLUSIONS Knowledge of distinctive assay performance characteristics of commercially available qPCR reagent mixes is critical for carefully designing analytical assay systems. The ABI, ABgene and Eppendorf reagent systems performed consistently overall for the two TaqMan assays for B. anthracis detection that were used in the current study. However, the use of Eppendorf reagent system requires shorter thermal cycling time. In addition, while the ABI and Eppendorf systems have similar assay sensitivity for both the rpoB and pag assays, the Eppendorf system achieves the same with lower C(T) values.

Rapid Expression of Mycobacterium avium subsp. paratuberculosis Recombinant Proteins for Antigen Discovery

ABSTRACT Mycobacterium avium subsp. paratuberculosis is the causative agent of Johnes disease, a chronic granulomatous enteritis of ruminants and other species. Detection of infection in animals is hampered by the lack of sensitive and specific diagnostic assays. We describe here an approach that utilizes translationally active PCR fragments for the rapid in vitro transcription and translation of recombinant proteins for antigen discovery in M. avium subsp. paratuberculosis. The investigations showed that the MAP1272c protein selectively reacts with sera from Johnes disease-positive cattle and represents an antigen of potential utility in M. avium subsp. paratuberculosis immunodiagnostics.

Mycobacterium avium subspecies paratuberculosis

Mycobacterium avium subspecies paratuberculosis, the causative agent of Johne’s disease, is distributed worldwide in farmed ruminant animals such as cattle, sheep and goats, and in wildlife such as rabbits, deer, antelopes, and bison. The major impact of this disease is on the worlds milk industry. When the focus is on a particular mutant phenotype such as attenuation in cultured macrophages or in mice, then some defined virulence determinants have recently been identified. The key genomic attributes of the bovine isolate of M. aviumsubsp. paratuberculosis, designated strain K-10, are discussed in this chapter. The chapter provides some applications of genome sequences. None of the studies have been applied genomic tools to directly address the role of M. avium subsp. One strain typing study examined caprine isolates of M. avium subsp. paratuberculosis using three molecular techniques: PFGE, IS900 RFLP analysis and IS1311 PCR-restriction enzyme analysis. This study found that PFGE analysis was more discriminatory than the other two methods, enabling a resolution of 13 different PFGE profiles among the 44 isolates evaluated. Researchers have also investigated this method but found more consistent expression of M. avium subsp. paratuberculosisproteins using the traditional recombinant protein production approach.

Assessment of sperm functional competence and sperm-egg interaction.

A precise understanding in the functional competence of mammalian sperm is essential to generate clinical advances for the treatment of infertility and novel contraceptive strategies. The fundamental knowledge on the controlling parameters for spermatozoal activation process will help in the identifying the causes in fertilization failure due to male factor as well as in developing male contraceptive methodologies. The defects in the sperm-egg interaction seem to be one of the controlling mechanisms, however, none of the presently available methods for the evaluation of the fertilizing ability of sperm precisely indicates the reason for the failure or the success of sperm entry into egg. Adequate number of motile spermatozoa with normal morphology and timely occurrence of acrosome reaction are presumably the major prerequisites for the penetration through the egg investments. The present communication briefly reviews some of the main features of mammalian sperm which control the success or the failure of fertilization and existing clinical methods indicating the lack of fundamental knowledge on the sub-cellular and molecular aspects of this unique and species-specific cell-cell interaction.

Cloning and development of synthetic internal amplification control for Bacillus anthracis real-time polymerase chain reaction assays.

An internal amplification control (IAC) was developed for Bacillus anthracis rpoB gene detection using TaqMan assay. Synthetic IAC oligonucleotides were subcloned using vector pDG1730 for ectopic integration into host Bacillus subtilis strain 1A772 genome. Differentially labeled target and IAC probes were used in real-time polymerase chain reaction (PCR) assays. There was no nonspecific cross-detection in single-well reactions. Limit of detection for both target and IAC DNA was 5 fg corresponding to a single gene copy. The IAC, in conjunction with target system, should decrease the rate of false-positive and false-negative results in real-time PCR assays.