Virus isolation vs RT-PCR: which method is more successful in detecting VHSV and IHNV in fish tissue sampled under field conditions?

This study compared the results of reverse transcription-polymerase chain reaction (RT-PCR) and traditional virus isolation on cell culture in detection of viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV). RT-PCR was used for 172 tissue sample pools (total of 859 fish) originating from a field survey on the occurrence of VHSV and IHNV in farmed and wild salmonids in Switzerland. These samples represented all sites with fish that were either identified as virus-positive by means of virus isolation (three sites, four positive tissue sample pools) and/or demonstrated positive anti-VHSV-antibody titres (83 sites, 121 positive blood samples) in a serum plaque neutralization test (SPNT). The RT-PCR technique confirmed the four VHSV-positive tissue sample pools detected by virus isolation and additionally identified one VHSV-positive sample that showed positive anti-VHSV-AB titres, but was negative in virus isolation. With IHNV, RT-PCR detected two positive samples not identified by virus isolation while in these fish the SPNT result had been questionable. One of the IHNV-positive samples represents the first detection of IHNV-RNA in wild brown trout in Switzerland. Compared to SPNT, the RT-PCR method detected, as with virus isolation, a much lower number of positive cases; reasons for this discrepancy are discussed. Our results indicate that RT-PCR can not only be successfully applied in field surveys, but may also be slightly more sensitive than virus isolation. However, in a titration experiment under laboratory conditions, the sensitivity of RT-PCR was not significantly higher when compared with virus isolation.

Selecting control genes for RT-QPCR using public microarray data

BACKGROUND: Gene expression analysis has emerged as a major biological research area, with real-time quantitative reverse transcription PCR (RT-QPCR) being one of the most accurate and widely used techniques for expression profiling of selected genes. In order to obtain results that are comparable across assays, a stable normalization strategy is required. In general, the normalization of PCR measurements between different samples uses one to several control genes (e.g. housekeeping genes), from which a baseline reference level is constructed. Thus, the choice of the control genes is of utmost importance, yet there is not a generally accepted standard technique for screening a large number of candidates and identifying the best ones. RESULTS: We propose a novel approach for scoring and ranking candidate genes for their suitability as control genes. Our approach relies on publicly available microarray data and allows the combination of multiple data sets originating from different platforms and/or representing different pathologies. The use of microarray data allows the screening of tens of thousands of genes, producing very comprehensive lists of candidates. We also provide two lists of candidate control genes: one which is breast cancer-specific and one with more general applicability. Two genes from the breast cancer list which had not been previously used as control genes are identified and validated by RT-QPCR. Open source R functions are available at http://www.isrec.isb-sib.ch/~vpopovic/research/ CONCLUSION: We proposed a new method for identifying candidate control genes for RT-QPCR which was able to rank thousands of genes according to some predefined suitability criteria and we applied it to the case of breast cancer. We also empirically showed that translating the results from microarray to PCR platform was achievable.

Molecular Surface Features in Modeling the HIV-1 RT Inhibitory Activity of 2-(2,6-Disubstituted phenyl)-3-(substituted pyrimidin-2-yl)-thiazolidin-4-ones

The human immunodeficiency virus-1 reverse transcriptase inhibitory activity of 2-(2,6-disubstituted phenyl)-3-(substituted pyrimidin-2-yl)-thiazolidin-4-ones have been analyzed using combinatorial protocol in multiple linear regression (CP-MLR) with several electronic and molecular surface area features of the compounds obtained from Molecular Operating Environment (MOE) software. The study has indicated the role of different charged molecular surface areas in modeling the inhibitory activity of the compounds. The derived models collectively suggested that the compounds should be compact without bulky substitutions on its peripheries for better HIV-1 RT inhibitory activity. It also emphasized the necessity of hydrophobicity and compact structural features for their activity. The scope of the descriptors identified for these analogues have been verified by extending the dataset with different 2-(disubstituted phenyl)-3-(substituted pyridin-2-yl)-thiazolidin-4-ones. The joint analysis of extended dataset highlighted the information content of identified descriptors in modeling the HIV-1 RT inhibitory activity of the compounds.

Real-Time RT-PCR for the Detection of Lyssavirus Species

The causative agents of rabies are single-stranded, negative-sense RNA viruses in the genus Lyssavirus of Rhabdoviridae, consisting of twelve classified and three as yet unclassified species including classical rabies virus (RABV). Highly neurotropic RABV causes rapidly progressive encephalomyelitis with nearly invariable fatal outcome. Rapid and reliable diagnosis of rabies is highly relevant for public and veterinary health. Due to growing variety of the genus Lyssavirus observed, the development of suitable molecular assays for diagnosis and differentiation is challenging. This work focused on the establishment of a suitable real-time RT-PCR technique for rabies diagnosis as a complement to fluorescent antibody test and rabies tissue culture infection test as gold standard for diagnosis and confirmation. The real-time RT-PCR was adapted with the goal to detect the whole spectrum of lyssavirus species, for nine of which synthesized DNA fragments were used. For the detection of species, seven probes were developed. Serial dilutions of the rabies virus strain CVS-11 showed a 100-fold higher sensitivity of real-time PCR compared to heminested RT-PCR. Using a panel of thirty-one lyssaviruses representing four species, the suitability of the protocol could be shown. Phylogenetic analysis of the sequences obtained by heminested PCR allowed correct classification of all viruses used.

Be-rêšît

Josef Hirschel

Ḥamiššā ḥumšê Tôrā : ʿim qeṣat pērûš Rašî ... we-gam hā-hafṭārôt ... we-5 megîllôt û-birkôt hat-Tôrā ...

[Jehuda Ben-Moses Naphtali]

Sēfer Menôrat ham-mā'ôr : we-ze maʿaśē ham-menôrā ʿim 7 nērôt ... ʿim haʿtāqā li-lšôn aškenaz

ḥibberô ... Yiṣḥāq Abôhab

Sēfer Menôrat ham-mā'ôr : we-ze maʿaśe ham-menôrā ʿim 7 nērôt ... ʿim haʿtāqā li-lšôn aškenaz ...

ḥibberô ... Yiṣḥāq Abôhab

Sēfer Menôrat ham-mā'ôr : we-ze maʿaśe ham-menôrā ʿim 7 nērôt ... li-lšôn aškenaz

Yiṣḥāq Abôhab

Sēfer Menôrat ham-mā'ôr : we-ze maʿaśē ham-menôrā ʿim 7 nērôt ... ʿim haʿtāqā li-lšôn aškenaz

ḥibbērô ... Yiṣḥāq Abôhab

Ḥamiššā ḥūmšê Tôrā, megillôt we-hafṭārôt bi-lšôn aškenaz : ṣe'ênā û-re'ênā benôt Ṣiyyôn bā-ʿaṭārā

še-ʿiṭṭēr lākem ... Yaʿaqôv Ben- ... Yiṣḥāq ... mim-mišpaḥat rabbēnû ... be-q"q Yānôw

Ḥamiššā ḥūmšê Tôrā, megillôt we-hafṭārôt bi-lšôn aškenaz : ṣe'ênā û-re'ênā benôt Ṣiyyôn bā-ʿaṭārā

še-ʿiṭṭēr lākem ... Yaʿaqôv Ben- ... Yiṣḥāq ... mim-mišpaḥat rabbēnû ... be-q"q Yānôw