{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T12:06:50Z","timestamp":1791806810442,"version":"4.3.4"},"reference-count":32,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2022,6,20]],"date-time":"2022-06-20T00:00:00Z","timestamp":1655683200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Berry College"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Tick-borne infections are a serious threat to humans, livestock, and companion animals in many parts of the world, often leading to high morbidity and mortality rates, along with decreased production values and\/or costly treatments. The prevalence of the microbes responsible for these infections is typically assessed by the molecular identification of pathogens within the tick vectors. Ticks sampled from animals are often engorged with animal blood, presenting difficulties in the amplification of nucleic acids due to the inhibitory effects of mammalian blood on the enzymes used in polymerase chain reactions (PCRs). This study tested two tick preparation methods, three methods of DNA extraction, and four commercially available DNA polymerases to determine the most reliable method of extracting and amplifying DNA from engorged ticks. Our study found that the phenol\u2013chloroform extraction method yielded the highest concentration of DNA, yet DNA extracted by this method was amplified the least successfully. Thermo Scientific\u2019s Phusion Plus PCR Master Mix was the best at amplifying the tick 16s rRNA gene, regardless of extraction method. Finally, our study identified that using the Qiagen DNeasy Blood &amp; Tissues kit for DNA extraction coupled with either Phusion Plus PCR Master Mix or GoTaq DNA polymerase Master Mix is the best combination for the optimized amplification of DNA extracted from engorged ticks.<\/jats:p>","DOI":"10.3390\/microorganisms10061254","type":"journal-article","created":{"date-parts":[[2022,6,21]],"date-time":"2022-06-21T04:39:55Z","timestamp":1655786395000},"page":"1254","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":22,"title":["Comparison of DNA Extraction and Amplification Techniques for Use with Engorged Hard-Bodied Ticks"],"prefix":"10.3390","volume":"10","author":[{"given":"Gretchen C.","family":"Reifenberger","sequence":"first","affiliation":[{"name":"Department of Biology, Berry College, Mount Berry, GA 30149, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bryce A.","family":"Thomas","sequence":"additional","affiliation":[{"name":"Department of Biology, Berry College, Mount Berry, GA 30149, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1945-3360","authenticated-orcid":false,"given":"DeLacy V. L.","family":"Rhodes","sequence":"additional","affiliation":[{"name":"Department of Biology, Berry College, Mount Berry, GA 30149, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,6,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"301","DOI":"10.14202\/vetworld.2015.301-315","article-title":"Tick-Borne Infections in Human and Animal Population Worldwide","volume":"8","author":"Duarte","year":"2015","journal-title":"Vet. World"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"781","DOI":"10.1099\/jmm.0.001206","article-title":"Emerging Tick-Borne Pathogens of Public Health Importance: A Mini-Review","volume":"69","author":"Rochlin","year":"2020","journal-title":"J. Med. Microbiol."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Wikel, S.K. (2018). Ticks and Tick-Borne Infections: Complex Ecology, Agents, and Host Interactions. Vet. Sci., 5.","DOI":"10.3390\/vetsci5020060"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1007\/BF01219108","article-title":"A Comparison of Methods for Sampling the Deer Tick, Ixodes dammini, in a Lyme Disease Endemic Area","volume":"14","author":"Falco","year":"1992","journal-title":"Exp. Appl. Acarol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"692","DOI":"10.1093\/jme\/tjaa207","article-title":"A Comparison of Tick Collection Materials and Methods in Southeastern Virginia","volume":"58","author":"Espada","year":"2021","journal-title":"J. Med. Entomol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1007\/s10493-013-9671-0","article-title":"Efficiency of Flagging and Dragging for Tick Collection","volume":"61","author":"Lia","year":"2013","journal-title":"Exp. Appl. Acarol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"473","DOI":"10.1007\/BF00048266","article-title":"Validation and Rapid Extraction of Nucleic Acids from Alcohol-Preserved Ticks","volume":"19","author":"Hubbard","year":"1995","journal-title":"Exp. Appl. Acarol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"e1147","DOI":"10.7717\/peerj.1147","article-title":"To Beat or Not to Beat a Tick: Comparison of DNA Extraction Methods for Ticks (Ixodes scapularis)","volume":"3","author":"Ammazzalorso","year":"2015","journal-title":"PeerJ"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"709","DOI":"10.1051\/vetres:2004038","article-title":"Determination of an Efficient and Reliable Method for DNA Extraction from Ticks","volume":"35","author":"Halos","year":"2004","journal-title":"Vet. Res."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"725","DOI":"10.1051\/vetres:2006031","article-title":"Comparison of Four Methods of Extracting DNA from D. gallinae (Acari: Dermanyssidae)","volume":"37","author":"Desloire","year":"2006","journal-title":"Vet. Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"592","DOI":"10.1186\/s13071-015-1185-7","article-title":"Insights into the Development of Ixodes scapularis: A Resource for Research on a Medically Important Tick Species","volume":"8","author":"Kocan","year":"2015","journal-title":"Parasites Vectors"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"339","DOI":"10.4269\/ajtmh.1997.56.339","article-title":"Inhibition of Efficient Polymerase Chain Reaction Amplification of Borrelia burgdorferi DNA in Blood-Fed Ticks","volume":"56","author":"Schwartz","year":"1997","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2569","DOI":"10.1007\/s00216-018-0931-z","article-title":"Inhibition Mechanisms of Hemoglobin, Immunoglobulin G, and Whole Blood in Digital and Real-Time PCR","volume":"410","author":"Sidstedt","year":"2018","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"485","DOI":"10.1128\/JCM.39.2.485-493.2001","article-title":"Purification and Characterization of PCR-Inhibitory Components in Blood Cells","volume":"39","year":"2001","journal-title":"J. Clin. Microbiol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1111\/j.1365-2915.2010.00903.x","article-title":"Evaluating Levels of PCR Efficiency and Genotyping Error in DNA Extracted from Engorged and Non-Engorged Female Dermacentor variabilis Ticks","volume":"25","author":"Dharmarajan","year":"2011","journal-title":"Med. Vet. Entomol."},{"key":"ref_16","first-page":"223","article-title":"Determination of an Efficient and Reliable Method for PCR Detection of Borrelial DNA from Engorged Ticks","volume":"10","author":"Stroil","year":"2021","journal-title":"Persian J. Acarol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"361","DOI":"10.1006\/mpev.1995.1033","article-title":"Discriminating between Ixodes Ticks by Means of Mitochondrial DNA Sequences","volume":"4","author":"Caporale","year":"1995","journal-title":"Mol. Phylogenetics Evol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"335","DOI":"10.1007\/s10493-009-9331-6","article-title":"Hosts, Distribution and Genetic Divergence (16S RDNA) of Amblyomma dubitatum (Acari: Ixodidae)","volume":"51","author":"Nava","year":"2010","journal-title":"Exp. Appl. Acarol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1147","DOI":"10.3201\/eid1407.071599","article-title":"Anaplasma phagocytophilum Infection in Small Mammal Hosts of Ixodes Ticks, Western United States","volume":"14","author":"Foley","year":"2008","journal-title":"Emerg. Infect. Dis."},{"key":"ref_20","first-page":"13","article-title":"DNA Extraction and Quantitation of Forensic Samples Using the Phenol-Chloroform Method and Real-Time PCR","volume":"297","author":"Parson","year":"2005","journal-title":"Methods Mol. Biol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1","DOI":"10.3354\/dao02822","article-title":"Differential Efficiency among DNA Extraction Methods Influences Detection of the Amphibian Pathogen Batrachochytrium dendrobatidis","volume":"113","author":"Bletz","year":"2015","journal-title":"Dis. Aquat. Org."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2058","DOI":"10.1128\/jcm.34.9.2058-2065.1996","article-title":"Differentiation of Spotted Fever Group Rickettsiae by Sequencing and Analysis of Restriction Fragment Length Polymorphism of PCR-Amplified DNA of the Gene Encoding the Protein r-OmpA","volume":"34","author":"Roux","year":"1996","journal-title":"J. Clin. Microbiol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1093\/jmedent\/32.3.213","article-title":"Use of Polymerase Chain Reaction to Detect Bacteria in Arthropods: A Review","volume":"32","author":"Higgins","year":"1995","journal-title":"J. Med. Entomol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"4396","DOI":"10.1128\/IAI.00393-09","article-title":"Comparative Analysis of the Properties and Ligand Binding Characteristics of CspZ, a Factor H Binding Protein, Derived from Borrelia burgdorferi Isolates of Human Origin","volume":"77","author":"Rogers","year":"2009","journal-title":"Infect. Immun."},{"key":"ref_25","unstructured":"(2022, May 12). Ops Diagnostics. Available online: https:\/\/opsdiagnostics.com\/notes\/protocols\/spin_column_plant_protocol.html."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Stanley, H., and Rhodes, D.V.L. (2021). Presence of Rickettsia Species in Ticks Collected from Companion Animals in Northeastern Georgia, United States. Vet. Sci., 8.","DOI":"10.3390\/vetsci8030037"},{"key":"ref_27","unstructured":"R Core Team (2021). R: A Language and Environment for Statistical Computing, R Foundation for Statistical Computing."},{"key":"ref_28","first-page":"1","article-title":"Efficiency and Reliability of DNA Extraction Methods for Ixodid Ticks (Acari: Ixodidae): A Comparative Tour","volume":"5","author":"Negi","year":"2019","journal-title":"Int. J. Anim. Biotechnol. Appl."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1186\/s41935-018-0065-7","article-title":"Concentration and Purity DNA Spectrophotometer: Sodium Monofluorophosphate Forensic Impended Effect","volume":"8","author":"Nzilibili","year":"2018","journal-title":"Egypt. J. Forensic Sci."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1465","DOI":"10.1016\/j.sjbs.2016.09.016","article-title":"Optimization of Conditions to Extract High Quality DNA for PCR Analysis from Whole Blood Using SDS-Proteinase K Method","volume":"24","author":"Qamar","year":"2017","journal-title":"Saudi. J. Biol. Sci."},{"key":"ref_31","unstructured":"(2022, May 11). PhusionTM Plus PCR Master Mix. Available online: https:\/\/www.thermofisher.com\/order\/catalog\/product\/F631XL."},{"key":"ref_32","unstructured":"(2022, May 11). Hemo KlenTaq\u00ae|NEB. Available online: https:\/\/www.neb.com\/products\/m0332-hemo-klentaq#Product%20Information."}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/10\/6\/1254\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T23:36:04Z","timestamp":1760139364000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/10\/6\/1254"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,6,20]]},"references-count":32,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2022,6]]}},"alternative-id":["microorganisms10061254"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms10061254","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,6,20]]}}}