Detection of Nosema bombycis by FTA Cards and Loop-Mediated Isothermal Amplification (LAMP)

Abstract

We successfully established a detection method which exhibited a markedly higher sensitivity than previously developed detection methods for Nosema bombycis by combining glass beads, FTA card, and LAMP. Spores of N. bombycis were first broken by acid-washed glass beads; the DNA was subsequently extracted and purified with the FTA card, and LAMP was performed using primers (LSU296) designed based on the sequence of the LSU rRNA of N. bombycis. The minimum detection concentration was 10 spores/mL. When this method was used to detect pebrine disease in silkworm egg, the detection rate for 500 silkworm eggs, in which only one egg was infected with N. bombycis, was 100 % under our optimized conditions. If the number of eggs in the sample increased to 800 or 1,000, the sample was divided into two equal portions, and the eggs were smashed with glass beads after the addition of 1 mL of TE buffer. The liquid in two tubes was later mixed and applied to the FTA card, and the detection rates were 100 %. Furthermore, the LAMP method established in our study could detect N. bombycis infection in silkworm 24 h earlier than microscopy.

Fast Technology Analysis Enables Identification of Species and Genotypes of Latent Microsporidia Infections in Healthy Native Cameroonians

“…; Yan et al. ). As verified in the present study, FTA cards are useful for preserving parasite DNA for subsequent amplification and sequencing and for safe transportation or shipment of specimens for subsequent testing.…”

Rapid and sensitive detection of Nosema bombycis using loop-mediated isothermal amplification and colorimetric nanogold

“…Purified microsporidian spores or silkworm eggs were placed in a 2 ml tube, and glass beads (Sigma Aldrich (Shanghai) Trading Co., Ltd., China) (1/2 of the volume of the tube) and 1 ml TE (10 mM tris-HCl and 1 mM EDTA, pH 8.0) buffer were added into the tube 4 . The sample was crushed by a Mini-Bead Beater (BioSpec, USA) at 4800g for 5 min with glass beads and buffer, and 100 µl of the resulting solution of broken spores was added to the FTA card (Whatman, UK).…”

“…Silkworm pebrine disease is a devastating disease in sericulture. N. bombycis cannot only parasitize in the silkworm larva, pupa, and moth, but can also be transmitted to the next generation through the silkworm eggs, which leads to a decline in the yield and quality of cocoons, and causes great losses in sericulture 4 . Because it is a kind of infectious disease and can be transmitted to the progeny through the eggs, silkworm pebrine disease is listed as the only inspection and quarantine target in silkworm eggs production.…”

“…Spot check of the female silkworm moths is the most important part of the pebrine inspection work, but its accuracy is affected by many factors, which may cause unqualified silkworm eggs to flow into the market and would make the users suffer unnecessary economic losses 4,5 . Many methods such as identification by naked-eyes, microscopic examination, serological and molecular biology detection techniques, were established to detect the N. bombycis [6][7][8] .…”

“…Many methods such as identification by naked-eyes, microscopic examination, serological and molecular biology detection techniques, were established to detect the N. bombycis [6][7][8] . To improve the detection of N. bombycis, China also has introduced the eggs testing for silkworm pebrine disease as a reserved inspection project 4 . However, the inspection for pebrine disease of silkworm eggs under microscopy is a time-consuming, labor-intensive work due to the huge amount of silkworm eggs.…”