Application of Pebrine Detection by PCR Infected <i>Bombyx Mori</i> Eggs

Abstract

With the development of the PCR technology, especially the improvement of its reagent and a method of pebrine detection by PCR in infected Bombyx mori eggs was established.With the 16sRNA gene of Nosema bombycis as target sequence, the results of extraction of genomic DNA from purified microspores showed that 1.3×10-7µg DNA can be extracted from each spore. The sensitivity detection showed the detection limit of Nosema bombycis DNA was 3.25×10-2pg, i.e. 4 spores. (PCR system volume is 25µl). The method of total DNA extraction from pebrine infected silkworm eggs just before hatching was created. The result showed that extracting total DNA from silkworm eggs after the eggs had been treated with 30% KOH met the PCR detection requirements. The result of application study showed the spores in the pebrine infected egg just before hatching can sensitively be found with PCR. The result of a group of eggs just before hatching detection showed that the maximum PCR detection level was of a pebrine infected eggs just before hatching in 300 healthy eggs when the total DNA extraction had been purified with Agarose electrophoresis. The probability of identifying groups of one pebrine spore in infected eggs just before hatching mixed with 100 healthy ones was about 80%.

Early and simultaneous detection ofNosema bombycis(Microsporidia: Nosematidae), nucleopolyhedrovirus (Baculoviridae), and densovirus (Parvoviridae) by multiplex real-time polymerase chain reaction inBombyx mori(Lepidoptera: Bombycidae)

“…BmNPV, Bombyx mori nucleopolyhedrovirus; BmDNV, Bombyx mori densovirus; + , positive result of the detection; − , no positive signal after 40 PCR cycles; N, negative controls. or liquid nitrogen freezing and grinding treatment before extraction, which breaks the spore walls and improves the DNA or protein yield (Xie 2007;Pan et al 2011). The recently developed method using a FastPrep agitator and glass beads has proven to be effective for DNA and protein extraction from microsporidian spores, fungi, and other microbial organisms with a thick and rigid outer wall (Fredricks et al 2005).…”

“…2010; Pan et al . 2011). In addition, several multiprimer PCRs were developed for the detection of microsporidian infections in B. mori (Hatakeyama and Hayasaka 2001, 2003).…”

“…In contrast, polymerase chain reaction (PCR)-based assays have undergone rapid development, and the use of this technique has become increasingly prevalent due to its simplicity, speed, specificity, and sensitivity. Some PCR-based approaches have been attempted for the detection of N. bombycis, NPV, and DNV, and some of these assays are well established (Kawakami et al 2001;Chaivisuthangkura et al 2009;Nimitphak et al 2010;Pan et al 2011). In addition, several multiprimer PCRs were developed for the detection of microsporidian infections in B. mori Hayasaka 2001, 2003).…”

Pebrine diagnosis using quantitative phase imaging and machine learning

“…Apart from these, other approaches for manual pebrine diagnosis such as dipstick, staining and delayed mother moth examination methods have been reported in literature 3, 12 . Sophisticated approaches which employ molecular diagnostic techniques such as polymerase chain reaction (PCR) 13–15 and fluorescent antibody method 16 are also explored. However, they are often constrained by their reliance on skilled workers, sophisticated equipment and sluggish sample preparation procedures.…”