Department of Recombinant Vaccines, Intercollegiate Faculty of Biotechnology University of Gdansk and Medical University of Gdansk, 80-807 Gdansk, Poland.
Embrapa Recursos Genéticos e Biotecnologia, Parque Estacao Biológica, 70770-900 Brasilia, Brazil.
Int J Mol Sci. 2017 Dec 28;19(1):83. doi: 10.3390/ijms19010083.
Baculoviruses have been used as biopesticides for decades. Recently, due to the excessive use of chemical pesticides there is a need for finding new agents that may be useful in biological protection. Sometimes few isolates or species are discovered in one host. In the past few years, many new baculovirus species have been isolated from environmental samples, thoroughly characterized and thanks to next generation sequencing methods their genomes are being deposited in the GenBank database. Next generation sequencing (NGS) methodology is the most certain way of detection, but it has many disadvantages. During our studies, we have developed a method based on Polymerase chain reaction (PCR) followed by Multitemperature Single Stranded Conformational Polymorphism (MSSCP) which allows for distinguishing new granulovirus isolates in only a few hours and at low-cost. On the basis of phylogenetic analysis of betabaculoviruses, representative species have been chosen. The alignment of highly conserved genes- and , was performed and the degenerate primers were designed to amplify the most variable, short DNA fragments flanked with the most conserved sequences. Afterwards, products of PCR reaction were analysed by MSSCP technique. In our opinion, the proposed method may be used for screening of new isolates derived from environmental samples.
杆状病毒已被用作生物农药数十年。最近，由于化学农药的过度使用，人们需要寻找新的可能有助于生物保护的药剂。有时在一种宿主中仅发现少数几个分离株或物种。在过去的几年中，已经从环境样本中分离出了许多新的杆状病毒物种，并对其进行了彻底的特征描述，并且由于新一代测序方法，它们的基因组被存入 GenBank 数据库。下一代测序（NGS）方法是最确定的检测方法，但它有许多缺点。在我们的研究中，我们开发了一种基于聚合酶链反应（PCR）的方法，随后是多温度单链构象多态性（MSSCP），它可以在数小时内且以低成本来区分新的颗粒病毒分离株。根据β杆状病毒的系统发育分析，选择了有代表性的物种。对高度保守的基因和进行了比对，并设计了简并引物来扩增侧翼序列最保守的、短的 DNA 片段。然后，通过 MSSCP 技术分析 PCR 反应产物。我们认为，所提出的方法可用于筛选源自环境样本的新分离株。
