Development and Evaluation of Taxon-Specific Primers for The
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Virus Research 263 (2019) 184–188 Contents lists available at ScienceDirect Virus Research journal homepage: www.elsevier.com/locate/virusres Short communication Development and evaluation of taxon-specific primers for the selected Caudovirales taxa T ⁎ Sandeep K. Newasea,b, Alka Guptac, Syed G. Dastagerd, Balu P. Kapadnisa, , ⁎ Ravindranath Shashidharb, a Department of Microbiology, Savitribai Phule Pune University, Ganeshkhind, Pune, 411007, India b Food Technology Division, Bhabha Atomic Research Centre, Mumbai, 400085, India c Molecular Biology Division, Bhabha Atomic Research Center, Mumbai, 400085, India d National Collection of Industrial Micro-organisms (NCIM) Resource Center, Biochemical Sciences Division, CSIR-NCL, Pune, 411008, India ARTICLE INFO ABSTRACT Keywords: The phage taxonomy is primarily based on the morphology derived from Transmission Electron Microscopic Bacteriophage (TEM) studies. TEM based characterization is authentic and accepted by scientific community. However, TEM Taxonomy based identification is expensive and time consuming. After the phage isolation, before analysis TEM, a DNA PCR based rapid method could be introduced. The DNA based method could dramatically reduce the number of TEM samples analyzed by TEM and thereby increase the speed and reduce the cost of identification. In the present DNA markers work, four environmental phage isolates were identified based on TEM studies and genome size. The identifi- Primers cation of these four phages was validated using DNA based method. The taxon-specific DNA markers were identified through multiple sequence alignments. The primers were designed at conserved genes (DNA poly- merase or integrase) of 4 different phage taxa viz. family Ackermannviridae, genus Jerseyvirus, genus T4virus, and genus P22virus. These primers were evaluated using both in vitro and in silico approach for the amplification of the target taxons. Majority of the primer sets were found to amplify member species of the targeted taxa in vitro. In In silico analysis, six primer sets intended for identification of family Ackermannviridae showed positive am- plification of ≥86.7% classified species. Further, the primers targeting the genus Jerseyvirus and T4virus showed the amplification of 53.8% and ≥84.6% species, respectively. The present work is a case study performed to explore the possibility of use of taxon-specific primers for identification and taxonomic studies of newly isolated phages to supplement the TEM. In the era of antimicrobial resistance, the researchers are in con- to get the quality images (Eskelinen, 2008; Williams and Carter, 1996). tinuous search of the phages which can be used as the alternatives to In certain instances, the solitary reliance on TEM observations has led the antibiotics. However, before such application, the collection of to anomalous phage taxonomy (Nelson, 2004). Till today there are no precise physiological and taxonomic information of the phages is cru- alternative methods to perform the taxonomic studies on phages be- cial, as there are safety concerns linked with the use of lysogenic phages sides TEM (Ackermann, 2013). (de Melo et al., 2018). The number of phage genomes has been increased considerably due The Transmission Electron Microscopy (TEM) is most notably used to current developments in Next-Generation Sequencing (NGS) tech- technique for bacteriophage (phage) taxonomy. This technique has nologies. With the leverage of ever-increasing phage genome data been used conservatively for morphological characterization and the available at biological databases, the signature genes can be identified. taxonomy of the phages since its invention (Ackermann, 2012, 2007; Further, the signature genes may be amplified, sequenced and analyzed Ackermann and Prangishvili, 2012; Aziz et al., 2018). TEM is termed as for the establishment of phage taxon (Clokie, 2009; Vallota-Eastman, “catch-all methods” in virus taxonomy due to its broad application and 2017). This approach is reliable and progressively getting established. availability of the results for quick assessment (Vale et al., 2010). PCR and its variants of have been used in clinical diagnosis, diversity However, TEM is expensive and time-consuming as the researcher has studies and detection of the viruses. The successful attempts have been to prepare the phage sample trickily and examine several sample areas made for the identification of phages by using the (Polymerase Chain ⁎ Corresponding authors. E-mail addresses: [email protected] (B.P. Kapadnis), [email protected] (R. Shashidhar). https://doi.org/10.1016/j.virusres.2019.02.005 Received 4 September 2018; Received in revised form 16 January 2019; Accepted 11 February 2019 Available online 12 February 2019 0168-1702/ © 2019 Elsevier B.V. All rights reserved. S.K. Newase, et al. Virus Research 263 (2019) 184–188 Reaction) PCR targeting several phage genes. DNA polymerase (gp43) using Primer-BLAST (Ye et al., 2012) (Performed 15 September 2018) and major capsid protein (gp23 and gp20) genes have been targeted to against RefSeq representative viral genomes (Brister et al., 2015)of score the diversity of the marine viruses and cyanophages (Clokie, order Caudovirales (NCBI:txid28883) with the default parameters. 2009). Primers based on the genes encoding for major capsid protein, Moreover, the primers were tested at insilico.ehu.eus/PCR/ (Bikandi tail tube protein, base plate wedge subunit and DNA polymerase have et al., 2004) for amplification of DNA targets of taxon members. Each been previously used for the phage identification (Anand et al., 2018; primer set was tested by allowing 0, 1 and 2 mismatches. The mis- Augustine et al., 2013; Clokie, 2009; Jäckel et al., 2017). Integrase has matches were not allowed in 10 nucleotides at 3′ end of the primer. been used as a signature gene for detection of prophage-encoded toxin The morphology of phages was observed using TEM with respect to genes in bacteria and for the diversity assessment of temperate phages head, tail and the fine structures. Based on the TEM image analysis or prophages (Adriaenssens and Cowan, 2014; Balding et al., 2005; (Fig. 1) and genome size (Table S1) the phage isolates were assigned to Colavecchio et al., 2017; Casas et al., 2006; Dwivedi et al., 2012; Yang four different families and putative genera as follows: phage PM10, et al., 2017). The amplification of phage DNA using PCR and the se- family Ackermannviridae genus Cba120virus (Adriaenssens et al., 2018); quencing of the amplification products is expedient and may be used in phage PM8, family Siphoviridae genus Jerseyvirus (Anany et al., 2015); complementation with TEM. phage Alpha-a, family Myoviridae genus T4virus (Krupovic et al., 2016) Four environmental phage isolates namely Salmonella phage and phage PM43, family Podoviridae; genus P22virus (Maniloff and vB_SalM_PM10 (PM10), vB_SalS_PM8 (PM8), Alpha-a and vB_SalP_PM43 Ackermann, 1998) (Table S1, Fig. S1b). (PM43) were assigned to the families Ackermannviridae, Siphoviridae, The phage taxon-specific primers were designed by aligning the Myoviridae and Podoviridae, respectively, based on the TEM observa- multiple genomes of the classified phages available at GenBank (NCBI) tions. However, considering the limitations of the TEM, an attempt was database (Clark et al., 2016). In the present study, maximum possible made to develop taxon-specific DNA markers for the establishment of homologous gene sequences in respective phage genera were included selected phage taxa (genera) which can be used alongside TEM to for the alignment. Gene selection was carried out in such a way that, at support the taxonomy. Based on genome size, partial genome sequen- least 50% of the available gene sequences for each phage genera were cing and DNA homology analysis carried out with help of DNA markers, covered. The detailed DNA data mining led to few potential genes for the aforesaid phages were assigned to the genera Cba120virus, four different phage genera belonging to order Caudovirales. In genus Jerseyvirus, T4virus and P22virus, respectively. Cba120virus genes encoding–topoisomerase, DNA helicase, host lysis The above-mentioned phages were isolated from sewage water protein and DNA polymerase were found to suitable for primer design. samples and their purity was ensured (Newase et al., 2018). Further, In genera Jerseyvirus and T4virus– gene DNA polymerase and in genus the phages were propagated (Carey-Smith et al., 2006) and con- P22virus– gene integrase were found to be appropriate for primer de- centrated (Bao et al., 2011) to obtain high titer stocks. The phage sign. Total 21 different primer sets with the average melting tempera- – particles from the stock (109–1010 PFU ml 1) were washed twice with ture of 50 °C were for four phage taxa were designed. The primers were 0.1 M ammonium acetate (pH 7.0) by centrifugation at 25,000 × g for evaluated in vitro against the phages which were identified based on 75 min. The final suspension was mixed with 2% phosphotungstate (pH comparative TEM genome analysis. For the genus Cba120virus,12 7.2) and placed on carbon-coated copper grid (Ted-Pella Inc. California, primer sets were tested, and five primers sets were found to be positive. USA). The grids were observed under LIBRA™ 120 TEM (Carl Zeiss, Primers for the Jerseyviruses (2 primer sets), T4viruses (3 primer sets) Oberkochen, Germany) operating at 120 kV. The head and tail mea- and P22viruses (2 primer sets) were tested. Two Jerseyvirus,3T4virus surements of the phage particles were taken using iTEM 5.1