Improving PCR and qPCR detection of hydrogenase A (hydA) associated with Clostridia in pure cultures and environmental sludges using bovine serum albumin

Detection of hydA genes of Clostridia spp. using degenerative and species specific primers for C. butyricum were optimized by the addition of bovine serum albumin (BSA) to polymerase chain reaction (PCR) and quantitative PCR (qPCR) reactions. BSA concentrations ranging from 100 to 400 ng/μl were examined using pure cultures and a variety of environmental samples as test targets. A BSA concentration of 100 ng/μl, which is lower than previously reported in the literature, was found to be most effective in improving the detection limit. The brightness of amplicons with 100 ng/μl BSA increased in ethidium bromide-treated gels, the minimum detection limit with BSA was at least one log greater, and cycle threshold (CT) values were lower than without BSA in qPCR indicating improved detection of target deoxyribonucleic acid for most samples tested. Although amplicon visualization was improved at BSA concentrations greater than or equal to 100 ng/μl, gene copy numbers detected by qPCR were less, CT values were increased, and Tm values were altered. SYBR Green dissociation curves of qPCR products of DNA from pure culture or sludge samples showed that BSA at 100 ng/μl reduced the variability of peak areas and Tm values.

[1]  Darrell P. Chandler,et al.  Reverse Transcriptase (RT) Inhibition of PCR at Low Concentrations of Template and Its Implications for Quantitative RT-PCR , 1998, Applied and Environmental Microbiology.

[2]  B. Olson,et al.  PCR-Restriction Fragment Length Polymorphism Method for Detection of Cyclospora cayetanensis in Environmental Waters without Microscopic Confirmation , 2003, Applied and Environmental Microbiology.

[3]  P. Rådström,et al.  Effects of Amplification Facilitators on Diagnostic PCR in the Presence of Blood, Feces, and Meat , 2000, Journal of Clinical Microbiology.

[4]  B H Olson,et al.  Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction , 1992, Applied and environmental microbiology.

[5]  X. Li,et al.  Attenuation of PCR inhibition in the presence of plant compounds by addition of BLOTTO , 1995, Nucleic Acids Res..

[6]  T. Fukushima,et al.  Runoff and loads of nutrients and heavy metals from an urbanized area. , 2006, Water science and technology : a journal of the International Association on Water Pollution Research.

[7]  J. Tiedje,et al.  DNA recovery from soils of diverse composition , 1996, Applied and environmental microbiology.

[8]  B H Olson,et al.  Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction , 1992, Applied and environmental microbiology.

[9]  J. Rose,et al.  Development of a PCR protocol for sensitive detection of Cryptosporidium oocysts in water samples , 1995, Applied and environmental microbiology.

[10]  J. Fuhrman,et al.  Rapid Detection of Enteroviruses in Small Volumes of Natural Waters by Real-Time Quantitative Reverse Transcriptase PCR , 2005, Applied and Environmental Microbiology.

[11]  David Moreira,et al.  Efficient removal of PCR inhibitors using agarose-embedded DNA preparations , 1998, Nucleic Acids Res..

[12]  Kevin E. Hicks,et al.  Substances interfering with direct detection of Mycobacterium tuberculosis in clinical specimens by PCR: effects of bovine serum albumin , 1996, Journal of clinical microbiology.

[13]  C. Saint,et al.  Development of a nested-PCR assay for the detection of Cryptosporidium parvum in finished water. , 2001, Water research.

[14]  Anne K Camper,et al.  Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. , 2006, Journal of microbiological methods.

[15]  Jo‐Shu Chang,et al.  Fermentative hydrogen production with Clostridium butyricum CGS5 isolated from anaerobic sewage sludge , 2005 .

[16]  R. L. Wolfe,et al.  Comparison of primers and optimization of PCR conditions for detection of Cryptosporidium parvum and Giardia lamblia in water , 1997, Applied and environmental microbiology.

[17]  J. Farrant,et al.  Inclusion of polyvinylpyrrolidone in the polymerase chain reaction reverses the inhibitory effects of polyphenolic contamination of RNA. , 1999, Nucleic acids research.

[18]  Y. Tsai,et al.  Rapid method for direct extraction of DNA from soil and sediments , 1991, Applied and environmental microbiology.

[19]  J. Werner,et al.  Copyright � 1995, American Society for Microbiology Inhibition of PCR by Aqueous and Vitreous Fluids , 1994 .

[20]  V. Maréchal,et al.  Quantification of enterovirus RNA in sludge samples using single tube real-time RT-PCR. , 2000, BioTechniques.

[21]  E. Galun,et al.  Comparison of methods for extraction of nucleic acid from hemolytic serum for PCR amplification of hepatitis B virus DNA sequences , 1997, Journal of clinical microbiology.

[22]  Donald E. Thompson,et al.  PCR detection of specific pathogens in water: a risk-based analysis. , 2002, Environmental science & technology.

[23]  K. Holmstrøm,et al.  Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions. , 1992, International journal of food microbiology.

[24]  Zhongtang Yu,et al.  Killing two birds with one stone: simultaneous extraction of DNA and RNA from activated sludge biomass , 1999 .

[25]  W. Vahjen,et al.  Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast , 1993, Applied and environmental microbiology.

[26]  A. Lew,et al.  PCR based diagnosis in the presence of 8% (v/v) blood. , 1991, Nucleic acids research.

[27]  K. Wilson.  Miniprep of bacterial genomic DNA , 1990 .

[28]  B. Swaminathan,et al.  Effect of ionic and nonionic detergents on the Taq polymerase. , 1990, BioTechniques.

[29]  T. Stinear,et al.  Detection of a single viable Cryptosporidium parvum oocyst in environmental water concentrates by reverse transcription-PCR , 1996, Applied and environmental microbiology.

[30]  R. Litaker,et al.  Rapid One-Step Quantitative Reverse Transcriptase PCR Assay with Competitive Internal Positive Control for Detection of Enteroviruses in Environmental Samples , 2006, Applied and Environmental Microbiology.

[31]  I G Wilson,et al.  Inhibition and facilitation of nucleic acid amplification , 1997, Applied and environmental microbiology.

[32]  Ajaib Singh,et al.  Development of Procedures for Direct Extraction of Cryptosporidium DNA from Water Concentrates and for Relief of PCR Inhibitors , 2005, Applied and Environmental Microbiology.

[33]  McKeown Bj.  An acetylated (nuclease-free) bovine serum albumin in a PCR buffer inhibits amplification , 1994 .

[34]  S. Tzipori,et al.  Parameters affecting polymerase chain reaction detection of waterborne Cryptosporidium parvum oocysts , 1997, Applied Microbiology and Biotechnology.

[35]  G. Widmer.  Genetic heterogeneity and PCR detection of Cryptosporidium parvum. , 1998, Advances in parasitology.

[36]  Anne K. Camper,et al.  Selective Removal of DNA from Dead Cells of Mixed Bacterial Communities by Use of Ethidium Monoazide , 2006, Applied and Environmental Microbiology.

[37]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[38]  C. Kreader.  Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein , 1996, Applied and environmental microbiology.