The development and application of a duplex reverse transcription loop-mediated isothermal amplification assay combined with a lateral flow dipstick method for Macrobrachium rosenbergii nodavirus and extra small virus isolated in China.

White tail disease (WTD), a major disease prevailing in the larval stage of Macrobrachium rosenbergii, caused by Macrobrachium rosenbergii nodavirus (MrNV) associated with extra small virus (XSV), led to the economic loss of shrimp industry in China. In order to establish a convenient, sensitive and selective molecular diagnostic method to detect MrNV and XSV for the Chinese shrimp (MrNV/XSV-chin), a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay combined with a lateral flow dipstick (LFD) method were developed. A set of four specific primers and a labeled probe were designed according to the six conserved gene sequence regions encoding for the MrNV capsid protein CP43 and the XSV capsid protein CP17. The detection of MrNV and XSV simultaneously by RT-LAMP was performed at 61 °C in a single reaction for 60 min followed by hybridization with an FITC-labeled probe for 5 min and visualized by LFD. The RT-LAMP-LFD assay had a sensitivity of approximately 100-fold higher than conventional PCR. In addition, the assay could detect MrNV/XSV-chin from limited amount of RNA extracts as low as 1.0 pg extracted from Macrobrachium rosenbergii. This assay was simple to use, required little instrumentation, and exhibited excellent specificity for the MrNV/XSV-chin compared with other shrimp viruses. In conclusion, a convenient, sensitive and selective practical molecular diagnostic method was developed with the potential for diagnosis and prevention of WTD.

[Rapid detection of Macrobrachium rosenbergii nodavirus isolated in China by a reverse-transcription loop-mediated isothermal amplification assay combined with a lateral flow dipstick method].

White coloration of the muscle of the giant river prawn (Macrobrachium rosenbergii) is a serious problem in China. The Macrobrachium rosenbergii Nodavirus (MrNV) has been confirmed to be the pathogen that causes this disorder. To develop a rapid, sensitive and specific technology for the detection of Macrobrachium rosenbergii Nodavirus isolated from China (MrNV-China), a reverse-transcription loop- mediated isothermal amplification assay combined with a lateral flow dipstick (RT-LAMP-LFD) assay method is described. A set of four primers and a labeled probe were designed specifically to recognize six distinct regions of the MrNV RNA2 gene. Results showed the sensitivity of the RT-LAMP-LFD assay was ten-times higher than the reverse-transcription loop-mediated isothermal amplification assay (RT-LAMP) with agarose gel electrophoresis. The assay was conducted with one-step amplification at 61°C in a single tube within 45 min. No product was generated from shrimps infected with other viruses, including DNA viruses (infectious hypodermal and hematopoietic necrosis virus (IHHNV); white spot syndrome virus (WSSV)) and RNA viruses (Taura syndrome virus (TSV); infectious myonecrosis virus (IMNV); yellow head virus (YHV)). Results were visualized by the LFD method. Therefore, the described rapid and sensitive assay is potentially useful for MrNV detection.

Development of a reverse transcription loop-mediated isothermal amplification assay for rapid detection of grass carp reovirus.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a gene amplification method that can amplify the RNA template by isothermal incubation. This paper reports a rapid and sensitive RT-LAMP method which was developed for the detection of grass carp reovirus (GCRV). The present study concluded the optimal conditions for the LAMP reaction of which the Mg(2+) concentrations in the reaction mixtures, the incubation temperature, and the reaction time are at 8mM, 64°C, and 30 min, respectively. The analytical sensitivity of the RT-LAMP method was revealed as low as 7 copies of viral templates and 100-fold more sensitive than the published RT-PCR method. A visual inspection of in-tube LAMP products stained with a DNA fluorescent dye demonstrated that the positive and negative reactions exhibit distinct and different colors in daylight, which means that gel electrophoresis is not necessary to judge the positive or negative results. As the application of the method is rapid, easy, and no complicated instrument required, the GCRV-RT-LAMP method established in this study has great potential for the detection of GCRV in both the laboratory and the farm.

Antiparasitic efficacy of dihydrosanguinarine and dihydrochelerythrine from Macleaya microcarpa against Ichthyophthirius multifiliis in richadsin (Squaliobarbus curriculus).

Ichthyophthirius multifiliis is a holotrichous protozoan that invades the gills and skin surfaces of fish and can cause morbidity and high mortality in most species of freshwater fish worldwide. The present study was undertaken to investigate the antiparasitic activity of crude extracts and pure compounds from the leaves of Macleaya microcarpa. The chloroform extract showed a promising antiparasitic activity against I. multifiliis. Based on these finding, the chloroform extract was fractionated on silica gel column chromatography in a bioactivity-guided isolation affording two compounds showing potent activity. The structures of the two compounds were elucidated as dihydrosanguinarine and dihydrochelerythrine by hydrogen and carbon-13 nuclear magnetic resonance spectrum and electron ionization mass spectrometry. The in vivo tests revealed that dihydrosanguinarine and dihydrochelerythrine were effective against I. multifiliis with median effective concentration (EC(50)) values of 5.18 and 9.43 mg/l, respectively. The acute toxicities (LC(50)) of dihydrosanguinarine and dihydrochelerythrine for richadsin were 13.3 and 18.2mg/l, respectively. The overall results provided important information for the potential application of dihydrosanguinarine and dihydrochelerythrine in the therapy of serious infection caused by I. multifiliis.

In vivo anthelmintic activity of chelidonine from Chelidonium majus L. against Dactylogyrus intermedius in Carassius auratus.

Dactylogyrus intermedius is one of the most common and serious cause of parasitic diseases of freshwater fish in aquaculture, and can cause morbidity and high mortality in most species of freshwater fish worldwide. To attempt controlling this parasite and explore novel potential antiparasitic agents, the present study was designed to ascertain the anthelmintic activity of Chelidonium majus L. whole plant and to isolate and characterize the active constituents against D. intermedius. The ethanol extract from C. majus whole plant showed significant anthelmintic activity against D. intermedius [EC(50) (median effective concentration) value = 71.5 mg L(-1)] and therefore subjected to further isolation and purification using various chromatographic techniques. A quaternary benzo[c]phenanthridine alkaloid exhibited significant activity against D. intermedius was obtained and identified as chelidonine. In vivo anthelmintic efficacy tests exhibited that chelidonine was 100% effective against D. intermedius at a concentration of 0.9 mg L(-1), with EC(50) value of 0.48 mg L(-1) after 48 h of exposure, which is more effective than the positive control, mebendazole (EC(50) value = 1.3 mg L(-1)). In addition, the 48-h median lethal concentration (LC(50)) for chelidonine against the host (Carassius auratus) was 4.54 mg L(-1). The resulting therapeutic index for chelidonine was 9.46. These results provided evidence that chelidonine might be potential sources of new antiparasitic drugs for the control of Dactylogyrus.

Effect of sanguinarine from the leaves of Macleaya cordata against Ichthyophthirius multifiliis in grass carp (Ctenopharyngodon idella).

The ciliate Ichthyophthirius multifiliis is one of the most pathogenic parasites of fish maintained in captivity. In this study, effects of crude extracts, fractions, and compounds from the leaves of Macleaya cordata against I. multifiliis were investigated under in vitro conditions by bioactivity-guided isolation method. The dried ethanol extract of M. cordata was extracted successively in a separating funnel with petroleum ether, ethyl acetate, chloroform and n-butanol. Among them, only the chloroform extract showed promising activity and therefore, was subjected to further separation and purification using various chromatographic techniques. Four compounds were isolated from chloroform extract, but only one compound showed potent activity. The structure of the active compound was elucidated as sanguinarine by hydrogen, carbon-13 nuclear magnetic resonance spectrum and electron ionization mass spectrometry. In vitro antiparasitic efficacy tests exhibited that sanguinarine was 100% effective against I. multifiliis at a concentration of 0.7 mg l(-1), with LC(50) and LC(90) values of 0.437 and 0.853 mg l(-1) after 4 h of exposure. In vivo antiparasitic efficacy tests showed that the number of I. multifiliis on the gills in the treatment group (in 0.9 mg l(-1) sanguinarine) was reduced by 96.8%, in comparison to untreated group at 25°C for 48 h. Mortality of fish did not occur in the treatment group during the trail, although 40% of untreated fish died. Our results indicate that the studied plant extracts, as well as sanguinarine might be potential sources of new antiparasitic drug for the control of I. multifiliis.

[Preparation and characterization of the mAb against Vibrio harveyi].

AIM: To prepare the mAb against Vibrio harveyi and analyse their biological properties. METHODS: 8-week-old female BALB/c mice were immunized with pathogenic V.harveyi GYC1108-1, which was isolated from the diseased great yellow croaker in Zhejiang China in 2003. Spleen cells collected from the immunized mice after the fifth immunization were fused with Sp2/0-Ag-14 myeloma cells. ELISA was used to screen hybridoma cells and identify the specificity and sensitivity of mAbs. RESULTS: Limited dilution method was used to subclone the positive clones. After three cycles of subcloning, 5 mAbs against GYC1108-1 were obtained and were proved to have high titer of ELISA. The test of specificity suggested that 1B7 reacted on V.harveyi, V.alginolyticus and V.parahaemolyticus but did not react on V.mimicus, V.anguillarum, Aeromonas hydrophila and the other isolates among the 14 bacterium species tested. 1B7 was used to detect V.harveyi at the concentration of 10(4) CFU/mL by indirect ELISA. CONCLUSION: The obtained mAbs could be used to diagnosis and prevent of V.harveyi in aquaculture rapidly.