Detection and identification of baculovirus pesticides by multitemperature single-strand conformational polymorphism

Abstract

The method of single-strand conformational polymorphism (SSCP) was modified in our laboratories for the characterization of baculoviruses, insect viruses with great potential for use as bioinsecticides in biological protection programs. A series of primers were synthesized after the comparison of the polyhedrin gene sequences of over 20 baculoviruses. Polyhedrin is a highly conserved protein which is responsible for the persistence of the virus in the environment. Universal primers were designed which could be used in polymerase chain reactions (PCR) containing genomic DNA from an array of nucleopolyhedrosis viruses (NPVs) including these which are used as biopesticides against important pests of forests and crops, such as Anticarsia gemmatalis, Spodoptera frugiperda, Lymantria dispar, Lymantria monacha and many others. PCR products were denatured and subjected to single-strand DNA electrophoresis at variable temperatures (MSSCP) where, after silver staining, they gave ssDNA band patterns characteristic for each baculovirus species. This technique can be potentially applied to detect baculoviruses in insects collected in the field, as well as to plant tissues and the excrements or bodies of predators without need for sequencing the PCR products. Sometimes MSSCP can be used not only for species determination but also as an indication of genomic variability which can be related to infectivity.

The sequence diversity and expression among genes of the folic acid biosynthesis pathway in industrial Saccharomyces strains

“…In the case of long pieces of sequence, a single mutation cannot raise the electrophoretic mobility shift, though conformation may change (Jordanova et al, 1997;Sunnucks et al, 2000). A substantial role in the efficacy of the method used is played by the temperature that significantly affects the stability and structure of the secondary DNA strand (Kaczanowski et al, 2001;Szewczyk et al, 2008;Tomczyk-Zak et al, 2012). Using changes of temperature during electrophoresis in 15-20% of the aforementioned studies increased the chance of distinction of two different conformations.…”

Analysis of Coinfections with A/H1N1 Strain Variants among Pigs in Poland by Multitemperature Single-Strand Conformational Polymorphism

“…It is an electrophoretic technique separating denatured PCR products according to their single-stranded secondary structure [ 32 , 33 ]. This method has been previously used for genetic characterization of a number of virus groups [ 58 – 60 ]. In our previous studies, MSSCP appeared to be very effective in detecting both mixed infections with different strains of IAV in humans and mutations in the gene regions responsible for the interaction with antibodies and evading the host immune response [ 61 , 62 ].…”

New Method for Differentiation of Granuloviruses (Betabaculoviruses) Based on Multitemperature Single Stranded Conformational Polymorphism

“…In the past, we described a method for detection and differentiation of NPVs [ 21 ]. In this paper, for the first time, we have proposed a method for discrimination of betabaculoviruses that can be used for screening new isolates/species of granuloviruses, which is based on the multitemperature single stranded conformational polymorphism (MSSCP) technique.…”