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Abstract: Mycoplasma suis cannot be cultivated in vitro. Therefore, PCR-based methods are irreplaceable for the diagnosis of M. suis infections especially when clinical symptoms are not evident. Currently, no easy and reliable method allowing the quantitative detection of M. suis is available. This report describes the development of a quantitative LightCycler™ PCR assay based on the msg1 gene of M. suis (LC MSG1 PCR). No PCR signals were obtained with closely related haemotrophic and non-haemotrophic mycoplasmas, with other bacteria, and with M. suis-free blood and tissue arguing for a high analytical specificity. Test sensitivity was found to be 100%, and test specificity 96.7%. To test the diagnostic suitability of the LC MSG1 PCR, 25 pigs with clinical porcine eperythrozoonosis and 25 healthy pigs were investigated. All ill pigs revealed a positive real-time PCR result whereas only one healthy pig was detected to be M. suis-infected. M. suis was quantitatively detected in 19 blood specimens of 100 sows from Switzerland and in 17 of 160 post-weaning piglets from Germany. In conclusion, this new LC MSG1 PCR assay represents a powerful tool for the improvement of the current M. suis diagnosis and for prevalence and pathogenesis studies. [Copyright &y& Elsevier]

Background: Herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2) and varicella-zoster virus (VZV) cause a wide range of signs and symptoms, varying from trivial mucocutaneous lesions to life-threatening infections, especially in immuno-suppressed patients. Since antiviral drugs are available, rapid and sensitive laboratory diagnosis of these virus infections is important. Objective: To set up and evaluate HSV-1, HSV-2 and VZV qualitative real-time PCR on the Lightcycler system and to compare the results with those of the ‘in-house’ nested PCR and virus isolation. Study design: 110 consecutive samples from dermal or genital lesions from patients suspected of having HSV infections and another 110 samples from patients with suspected VZV infections were tested with real-time PCR, nested PCR and virus isolation. Results: 24 samples (22%) were positive for HSV-1 by virus isolation and nested PCR, whereas 26 (24%) were positive by real-time PCR. HSV-2 was detected in 28 samples (25%) by virus isolation, in 41 (37%) by nested PCR and in 40 (36%) by real-time PCR. VZV was isolated in 15 samples (14%) and VZV DNA was detected in 51 samples (46%) by nested PCR as well as by real-time PCR. Nucleic acid amplification increased the detection rate of HSV-2 and VZV DNA in particular compared to virus isolation. No significant difference in sensitivity was found between real-time PCR and nested PCR. Conclusion: Real-time PCR has the advantage of rapid amplification, a reduced risk for contamination and it is a suitable method for diagnosis of VZV and HSV in specimens from skin lesions. [Copyright &y& Elsevier]

Porcine picornaviruses comprising at least 23 serotypes grouped into six species were described as causative agents of neurological disorders, reproductive failure, and aphthae-like dermal lesions of swine. Other viruses such as classical swine fever virus (CSFV), African swine fever virus, pseudorabies virus (PRV), vesicular stomatitis virus, vesicular exanthema virus, porcine respiratory and reproductive syndrome virus, and porcine parvovirus (PPV) may cause diseases with similar clinical symptoms. Therefore, rapid and reliable PCR detection of the most frequent porcine picornaviruses is of interest. A real-time RT-PCR protocol employing LightCycler technology to detect all known serotypes of the three porcine enterovirus (PEV) cytopathic effect (CPE) groups was established. It uses three sets of primer pairs and group-specific hybridisation probes. The primer pairs were designed to amplify highly conserved sequences of the 5′-non-translated region (5′-NTR) of the relevant virus species. The one-step real-time PCR based on the LightCycler technology is more rapid and less contamination-prone than the nested RT-PCR and allows the precise quantitation of the virus load in the tested specimens. All acknowledged serotypes of the three PEV CPE groups and all tested field strains isolated from clinical specimens were detectable. Viruses of the PEV CPE group III can be distinguished from the closely related swine vesicular disease virus (SVDV). [Copyright &y& Elsevier]

Abstract: Prior to PCR amplification, uracil-DNA glycosylase (UNG) can be incorporated to prevent amplicon contamination. Melting temperature (T m) analysis of herpes simplex virus (HSV) hybridization probes was used to demonstrate a concentration-dependent decrease in T m values when heat stabile or heat labile UNG was added. [Copyright &y& Elsevier]