A qPCR Targeted Against the Viral Replication Origin Designed to Quantify Total Amount of Filamentous Phages and Phagemids
Tóm tắt
Filamentous bacteriophages are widely used in phage display technology. The most common quantification method is lysis plaque formation test (PFT). This technique has several disadvantages, and only quantifies infective phages and is not effective when phagemids are used. We developed a qPCR method directed against the M13 replication origin, which detects between 3.3 × 103 and 3.3 × 108 viral genome copies with a linearity of R2 = 0.9998. Using this method we were able to observe a difference of approximately ten more phages than with the PFT. This difference was not due to the presence of a free genome, which suggests the presence of non-infective particles. Using a DNaseI treatment, we observed the presence of 30% to 40% of unpackaged genome in recombinant phage modified in PIII or PVIII. The qPCR method with a DNase I treatment is an efficient method to quantify the total amount of filamentous phages.
Tài liệu tham khảo
Smith GP (1985) Filamentous phage fusion: novel expression vectors that display cloned antigens on the surface of the virion. Science (80-) 228:1315–1317. https://doi.org/10.1126/science.4001944
Rakonjac J, Bennett NJ, Spagnuolo J et al (2011) Filamentous bacteriophage: biology, phage display and nanotechnology applications. Curr Issues Mol Biol 13:51–76. https://doi.org/10.1002/9780470015902.a0000777
Løset GÅ, Sandlie I (2012) Next generation phage display by use of pVII and pIX as display scaffolds. Methods 58:40–46. https://doi.org/10.1016/j.ymeth.2012.07.005
Henry KA, Arbabi-Ghahroudi M, Scott JK (2015) Beyond phage display: non-traditional applications of the filamentous bacteriophage as a vaccine carrier, therapeutic biologic, and bioconjugation scaffold. Front Microbiol 6:1–18. https://doi.org/10.3389/fmicb.2015.00755
Samoylova TI, Braden TD, Spencer JA, Bartol FF (2017) Immunocontraception: filamentous bacteriophage as a platform for vaccine development. Curr Med Chem 24:3907–3920. https://doi.org/10.2174/0929867324666170911160426
Rodi DJ, Makowski L (1999) Phage-display technology—finding a needle in a vast molecular haystack. Curr Opin Biotechnol 10:87–93. https://doi.org/10.1016/S0958-1669(99)80016-0
Olofsson L, Ankarloo J, Andersson PO, Nicholls IA (2001) Filamentous bacteriophage stability in non-aqueous media. Chem Biol 8:661–671. https://doi.org/10.1016/S1074-5521(01)00041-2
Tsuchiya H, Sawamura T, Harashima H, Kamiya H (2005) Correction of frameshift mutations with single-stranded and double-stranded DNA fragments prepared from phagemid/plasmid DNAs. Biol Pharm Bull 28:1958–1962
Russel M, Whirlow H, Sun TP, Webster RE (1988) Low-frequency infection of F-bacteria by transducing particles of filamentous bacteriophages. J Bacteriol 170:5312–5316. https://doi.org/10.1128/jb.170.11.5312-5316.1988
Szermer-Olearnik B, Drab M, Mąkosa M et al (2017) Aggregation/dispersion transitions of T4 phage triggered by environmental ion availability. J Nanobiotechnol 15:32. https://doi.org/10.1186/s12951-017-0266-5
Kłopot A, Zakrzewska A, Lecion D et al (2017) Real-time qPCR as a method for detection of antibody-neutralized phage particles. Front Microbiol 8:2170. https://doi.org/10.3389/fmicb.2017.02170
Edelman DC, Barletta J (2003) Real-time PCR provides improved detection and titer determination of bacteriophage. Biotechniques 35:368–375. https://doi.org/10.2144/03352rr02
Anderson B, Rashid MH, Carter C et al (2011) Enumeration of bacteriophage particles. Bacteriophage 1:86–93. https://doi.org/10.4161/bact.1.2.15456
Peng X, Nguyen A, Ghosh D (2018) Quantification of M13 and T7 bacteriophages by TaqMan and SYBR green qPCR. J Virol Methods 252:100–107. https://doi.org/10.1016/j.jviromet.2017.11.012
Guo YC, Zhou YF, Zhang XE et al (2006) Phage display mediated immuno-PCR. Nucl Acids Res 34:1–6. https://doi.org/10.1093/nar/gkl260
Yong SF, Ngeow YF, Tong YK, Ong JT (2006) Real-time PCR detection of male-specific coliphages. Malays J Pathol 28:79–82
Wan Z, Goddard NL (2012) Competition between conjugation and M13 phage infection in Escherichia coli in the absence of selection pressure: a kinetic study. Genes|Genomes|Genetics 2:1137–1144. https://doi.org/10.1534/g3.112.003418
Jaye DL, Nolte FS, Mazzucchelli L et al (2003) Use of real-time polymerase chain reaction to identify cell- and tissue-type-selective peptides by phage display. Am J Pathol 162:1419–1429. https://doi.org/10.1016/S0002-9440(10)64275-7
Untergasser A, Nijveen H, Rao X et al (2007) Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 35:W71–W74. https://doi.org/10.1093/nar/gkm306
Sambrook J, Russell DW (2006) Purification of nucleic acids by extraction with phenol: chloroform. CSH Protoc. https://doi.org/10.1101/pdb.prot4455
Rutledge RG (2003) Mathematics of quantitative kinetic PCR and the application of standard curves. Nucleic Acids Res 31:93e–93. https://doi.org/10.1093/nar/gng093
Reitinger S, Petriv OI, Mehr K et al (2012) Purification and quantitation of bacteriophage M13 using desalting spin columns and digital PCR. J Virol Methods 185:171–174. https://doi.org/10.1016/j.jviromet.2012.06.021
Cormier J, Janes M (2014) A double layer plaque assay using spread plate technique for enumeration of bacteriophage MS2. J Virol Methods 196:86–92. https://doi.org/10.1016/j.jviromet.2013.10.034
Gentilomi GA, Cricca M, De Luca G et al (2008) Rapid and sensitive detection of MS2 coliphages in wastewater samples by quantitative reverse transcriptase PCR. New Microbiol 31:273