UniversityCollege of SoutheastNorway Facultyof Arts and Sciences Institute of Environmentaland HealthStudies Bø in Telemark Master’sThesis Studyprogramme: Master of EnvironmentalScience. Spring20 16 NasirSultan In silico evaluation of PCR- pr imers for detection of Lyme Borrelia . University College of Southeast Norway Faculty of Arts and Sciences Institute of Environmental and Health Studies Bø in Telemark Gullbringvegen 36 3800 Bø http://www.usn.no © 2016 Nasir Sultan This thesis is worth 60 study points Cover-pictures Ixodex scapularis: pbase.com PCR: unlockinglifescode.org ___ 2 Abstract Lyme borreliosis (LB) or Lyme disease is the most prevalent vector-borne disease in US and Europe. The etiologic is some species of tick-borne spirochetes Borrelia burgdorferi sensu lato (B. burgdorferi sl) complex. The most common clinical symptoms of LB is the erythema migrans (EM). The pathogen is transmitted to humans through the tick bite of Ixodes species, and spread to cause more severe manifestations such as Acrodermatitis Chronica Atrophicans (ACA), Lyme arthritis, and neuroborreliosis. Although polymerase Chain reaction (PCR) assay is an important molecular analysis for detection of DNA-pathogen in infected organisms, an apparent discrepancy about its accuracy and reliability is still existing. In order to validate PCR assay for detection of LB, 77 PCR- primer pairs were assembled from previous publications, and investigated for specificity and sensitivity using bioinformatics applications. The primers targeted genes coding for outer surface proteins ospA (29), ospB (2), and ospC (11), flagellin flaB (25), outer membrane protein p66 (5), genetic recombination protein recA (2), and plasmid-specific sequence (3). Basic Local Alignment Search Tool (BLAST) was employed to search homology between sequence of primers and sequences of GenBank database to find out cross-reactivity with non-targeted taxa of Lyme Borrelia or relapsing fever Borrelia. The primer-set that showed similarity only for the targeted species was considered a specific primer-set, while the primer- set that homologous with untargeted Borrelia species was in cross-reaction and was considered unspecific for the target species or PCR analysis. The sensitivity of primer-sets was rational designed and presented by proportion of specific hits for particular species to the total hits number for the same species, which is diagnostic sensitivity (coverage). The results showed 25 (32%) specific primer-sets, 40 (52%) unspecific primer-sets, and 12 (16%) specific primer-sets but showed limited cross-reaction with untargeted Borrelia species. High proportion of ospA- primers were specific, most of flaB-primers were unspecific, and half of ospC-primers was specific. Most primers for remaining genes showed specificity for the species of interest. The vast majority of primer-sets ranged from low to moderate sensitive for the species of interest. This study demonstrated most PCR-primers that have been used previously for detection of Lyme Borrelia were suboptimal to be specific for taxon of interest and unsuitable to be specifically used in PCR for detection B. burgdorferi sl complex. ___ 3 Glossary and Abbreviations (Tick-borne spirochetes) - A spirochete bacteriae that are transmitted or carried by ticks. Algorithm - A fixed procedure embodied in a computer program. Alignment - The process or result of matching up the nucleotide or amino acid sequences of two or more biological sequences to achieve maximal levels of identity. Amplicon - The specific DNA product generated by PCR using one pair of PCR primers. DNA (deoxyribonucleic acid) - The genetic material of most living organisms, and consists of two complementary chains of nucleotides. Downloaded amplicon – A sequence of nucleotides was obtained in this master project. This sequence is containing the forward and reverse primers, and is corresponding the amplified segment of certain gene in the source reference of the primers. E value - The Expectation value or Expect value is the number of alignments with a particular score that are expected to occur by chance. Entrez - An integrated search and retrieval system that integrates information from various databases at NCBI, including nucleotide. Enzootic life cycle - A life cycle of microbial pathogen inside living organisms in an endemic area. Gap - A space introduced into an alignment to compensate for insertions and deletions in one sequence relative to another. GenBank - Primary nucleotide sequence database produced and maintained at the National Center for Biotechnology Information (NCBI). Gene - The unit of inheritance. Genes are sections of DNA which code for the production of a particular protein or protein subunit. Genome - The entire collection of genes in an organism. Hematophagous arthropod - Blood-feeding insects/ticks. ___ 4 Homocoel -The body cavity found in many invertebrates where the hemolymph circulates through. Homologous – two or more nucleotide sequences are similar or having a corresponding. Homology - A degree of similarity between two or more nucleotide sequences, as in position or structure, and that may indicate a common origin; a correspondence of structure. Host - A host organism is a living cell in which a microbial pathogen lives and reproduces using cell nourishment and components. In this study mammalians and rodents. Identity - The extent to which two nucleotide sequences have the same residues at the same positions in an alignment, often expressed as a percentage. MegaBLAST- A local pairwise nucleotide alignment tool that is optimized for finding long alignments between nearly identical sequences. Motif: A short conserved region in a nucleotide sequence. Motifs are frequently highly conserved parts of domains. Nucleotides - Are the building unit of DNA molecule and consisting of a sugar molecule (pentose), a phosphate group and one of four different bases: Adenine (A), Thymine (T), Cytosine (C) and Guanine (G). PCR: Polymerase Chain Reaction - A technique for amplifying DNA sequences in vitro by separating the DNA into two strands and incubating it with oligonucleotide primers and DNA polymerase. It can amplify a specific sequence of DNA by as many as one billion times Primer - Short synthetic single-stranded DNA fragment which is required to induce the synthesis of DNA in the PCR reaction. (Primer-pair) – The sequences of the individual primers (forward and reverse) without their downloaded amplicon. (Primer-set) – A primer set in this master thesis is presented by the forward and reverse primers and their corresponding downloaded amplicon. Query coverage (Qc)– The percentage that the query sequence is covered by the sequence of matched database. ___ 5 Query sequence - The input sequence (or other type of search term) to which all of the entries in a database are to be compared. Score (S) - A numerical value that describes the overall quality of an alignment or similarity between two or more sequences. Similarity - The extent to which nucleotide sequences are related. Similarity between two sequences can be expressed as percent sequence identity and/or percent positive substitutions. Taxon - A taxonomic group of any rank, such as a species, family, or class. Vector - A vector is an organism that carries / transmits amicrobial pathogen from among hosts. In this study are the ticks. Zoonotic disease - A disease can be spread from animals to humans. ACA- Acrodermatitis Chronica Atrophicans. BLAST - Basic Local Alignment Search Tool. CSF- Cerebrospinal fluid. CSF- Chronic fatigue syndrome. DDBJ - The DNA Data Bank of Japan. EM - Erythema Migrans. flaB- gene coding for flagellin B. LB - Lyme borreliosis. LD - Lyme disease. NCBI - The National Center for Biotechnology Information. Osp - outer surface protein. ospA, B. C - gene coding for outer surface protein A, B, C, respectively. p66 - gene coding for outer membrane protein. ___ 6 recA - gene recombination A. RFB - Relapsing fever Borrelia RMBL - The European Molecular Biology Laboratory. SF- synovial fluid. I. - Ixodes bp – base pair B. - Borrelia B.bu sl - B. burgdorferi sl - B. burgdorferi sensu lato. B.bu ss - B. burgdorferi ss - B. burgdorferi sensu stricto B.ga - B. garinii B.af- B. afzelii B.va - B. valaisiana B.spl - B. spielmanii B.bis - B. bissttii B.amr - B. americana B.bv - B. bavariensis B.jpn - B. japonica B.clf - B. californiensis B.fin - B. finlandensis B.chl - B. chilensis B.crl - B. carolinensis B.and - B. andersonii B.okn - B. okinawa B.lst - B. lusitaniae B.tnk - B. tanuki B.trd -B. turdii B.tur - B. turcica B.snc - B. sinica B.yng - B. yangtzensis B.dtn - B. duttonii ___ 7 B.prs - B. persica. B.crd - B. crocidurae. B.mty - B. miyamotoi. B.trc - B. turicatae. B.hsp - B. hispanica. B.rcnt - B. recurrentis B.hrm - B. hermsii B.prk - B. parkeri B.ans - B. anserina B.spp. - Undefined Borrelia species ___ 8 Contents Abstract ……………………………………………………………………………………………………………. 3 Glossary and Abbreviarion ..………………………………………………………………………………. 4 Foreword …………………………………………………………………………………………………………… 11 1 Introduction ........................................................................................................ 12 1.1 Borrelia .................................................................................................................. 12 1.1.1. Lyme disease Symptoms ............................................................................... 13 1.1.2. Lyme Borrelia Species ................................................................................... 14 1.1.3. Life Cycle .......................................................................................................