A review of nucleic-acid-based diagnostic tests for Babesia and Theileria, with emphasis on bovine piroplasms.

Nucleic acid-based methods offer a variety of tools for the detection of parasites. This field of veterinary and medical sciences is rapidly evolving, leading to greater sensitivity and higher throughput. One of the reasons justifying such a fast development is the fact that tests targeting several taxa can be created. The present article deals with the applications of molecular diagnostics of tick-borne diseases in Parasitology. Special attention is given to Babesia and Theileria species infecting livestock. The commonly used molecular methods in diagnostic of tick-transmitted hematic protozoa are the following: (i) final time polymerase chain reaction; (ii) reverse line blotting (RLB); (iii) real time PCR, based on SYBR Green or probe fluorescence; (iv) isothermal amplification methods: loop-mediated amplification (LAMP) and self sustaining sequence replication (3SR, also named as "Nucleic Acid Sequence Based Amplification", NASBA, or Transcription Mediated Amplification, TMA). In general, none of these methods could be considered better than another. Their score in diagnostic applications greatly depends on the laboratory size. Small-scale laboratories handling few samples may find final time PCR a cheap alternative. On the contrary, large-scale laboratories prefer methods amenable to automation, like RLB, PCR-ELISA or qPCR.

Detection of infectious salmon anaemia virus by real-time nucleic acid sequence based amplification.

We have developed a real-time nucleic acid sequence based amplification (NASBA) procedure for detection of infectious salmon anaemia virus (ISAV). Primers were designed to target a 124 nucleotide region of ISAV genome segment 8. Amplification products were detected in real-time with a molecular beacon (carboxyfluorescin [FAM]-labelled and methyl-red quenched) that recognised an internal region of the target amplicon. Amplification and detection were performed at 41 degrees C for 90 min in a Corbett Research Rotorgene. The real-time NASBA assay was compared to a conventional RT-PCR for ISAV detection. From a panel of 45 clinical samples, both assays detected ISAV in the same 19 samples. Based on the detection of a synthetic RNA target, the real-time NASBA procedure was approximately 100x more sensitive than conventional RT-PCR. These results suggest that real-time NASBA may represent a useful diagnostic procedure for ISAV.

Practical experiences of moving molecular diagnostics into routine use at the Veterinary Laboratories Agency.

The practical bench application of molecular tests (here defined as nucleic acid-based tests) in a routine testing situation is not without its challenges. This paper outlines the approaches we take at the Veterinary Laboratories Agency (VLA) and highlights some of the practical issues which we have found to be important for the successful introduction and use of molecular tests in a routine testing environment. The potential advantages of molecular tests, and factors which dictate which tests are adopted for routine testing, are discussed before giving some examples of molecular tests in routine use at the VLA. The instrumentation, reagents and assays we use are outlined, followed by sections of how we approach validation and how we manage and resource the transfer of molecular tests into routine use.