Loop-mediated isothermal amplification (LAMP) assay detected Salmonella within 60 min. The 220 strains of 39 serotypes of Salmonella subsp. enterica and 7 strains of Salmonella enterica subsp. arizonae were amplified, but not 62 strains of 23 bacterial species other than Salmonella. The sensitivity of the LAMP assay was found to be >2.2 cfu/test tube using nine serotypes. The specificity was similar to that of a PCR assay, but the sensitivity of LAMP was greater. Both fluorescence and turbidity were able to detect the products in the LAMP assay. S. enteritidis in a liquid egg sample artificially inoculated with the organism was detected by the LAMP assay at 2.8 cfu/test tube, although negative by PCR assay. These results indicate that the LAMP assay is a rapid, specific and sensitive detection method for Salmonella.

For establishment of a method for rapid diagnosis of Mycoplasma pneumoniae in clinical specimens, we designed and evaluated a loop-mediated isothermal amplification (LAMP) assay, using a set of primers targeting the SDC1 repetitive element of the M. pneumoniae genome. The method showed rapid and specific amplification for all the strains of M. pneumoniae tested (Type I and II), within 1 hour at 65 degrees C. No cross-reactivity with the most common causative organisms bacterial pneumonia was observed. The detection limit was shown as 6 copies, which was equal to or higher than that of the two nested PCRs used as references. Two hundred four clinical samples, comprising sputum samples, throat swabs, etc., were tested by both LAMP and PCR, and determination of the correlations revealed complete agreement individually (24 positives). The LAMP assay, a simple procedure in a closed system, allowed rapid amplification and accurate detection consistently; therefore we considered that it could become an caeasily available diagnostic method suitable for clinical situations requiring quick and appropriate decisions for treatments and care.

New methods were developed for the detection of koi herpesvirus (KHV, CyHV-3) by LAMP, which were compared with the PCR for specificity and sensitivity. We designed two primer sets targeting a specific sequence within the 9/5 PCR amplicon (9/5 LAMP) and the upper region of the SphI-5 PCR amplicon (SphI-5 LAMP), including a sequence highly conserved among the strains. The amplification was monitored in real-time based on the increase in turbidity, with magnesium pyrophosphate as the by-product. The reactions were carried out under isothermal conditions at 65• C for 60 min. The detection limit of both LAMP was six copies, equal to the modified SphI-5 PCR. No cross-reactivity with other fish pathogenic viruses and bacteria was observed. SphI-5 LAMP was found to have a quicker response in terms of the reaction velocity than 9/5 LAMP. Therefore, we consider SphI-5 LAMP to be superior for routine use. Additionally, LAMP was found applicable to crude extract from gills and other organs. LAMP methods are superior in terms of sensitivity, specificity, rapidity and simplicity, and are potentially a valuable diagnostic tool for KHV infections.

ABSTRACT-We designed a LAMP loop-mediated isothermal amplificatio primer set comprised with six primers from koi herpesvirus KHV, CyHV-3 specific PCR amplicon accession number: AF411803 and developed a rapid and sensitive detection method for KHV. The target sequence was amplified under isothermal condition at 65°C within 60 min, and the detection limit was at least six copies of the plasmid inserted KHV specific sequence. Cross-reactivity with other fish-pathogenic viruses and bacteria was not observed. The reaction was monitored in real-time by an increase in the turbidity of a large amount of by-product, pyrophosphate. As an alternative method, a visual detection at end-point of reaction could be achieved by an increase of turbidity in reaction mixture with naked eyes. The LAMP method was applicable to amplify the target sequence from crudely extracted samples of gills by a simple procedure.

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