use of pcr technology for the diagnostics of lyme disease€¦ · use of pcr technology for the...

Use of PCR technology for the diagnostics of Lyme disease

Recent developments in BCA-lab

Dr. Viktoria Krey, PhD

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Polymerase chain reaction

PCR = polymerase chain reaction Def: “PCR is an efficient and cost-effective method in molecular biology to enzymatically reproduce or ”amplify” a specific selected segment of DNA (RNA).”

Cycle 0 1

Cycle 1 2

Cycle 2 4

DNA polymerase

DNA polymerase

Exponential amplification of target sequence. Starting from 1 copy in the sample after 40 cycles: 240 = 1.099.511.628.000 copies

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PCR – functional principle

Components of a conventional PCR: • Thermostable DNA polymerase • Target DNA (e.g. isolated from host sample) • Oligonucleotide primers (sense and antisense primer) • dNTPs • MgCl2

• PCR buffer • Water

Thomas A. Steitz J. Biol. Chem. 1999;274:17395-17398

©1999 by American Society for Biochemistry and Molecular Biology

Phases of PCR:

Denaturation (separation of DNA double strand)

Annealing (hybridization of primer)

Elongation (extension of DNA strand)

Initial Denaturation

Final Elongation

Cycle

Phases of PCR:



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Phases of PCR:

Denaturation (95 °C)




Final Elongation

Cycle

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5’-CATGTGCATGACGCCTAATAGCGCGA-3’ I I I I I I I I I I I I I I I I I I I I I I I I I I 3’-GTACACGTACTGCGGATTATCGCGCT-5’

5’-CATGTGCATGACGCCTAATAGCGCGA-3’ I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 3’-GTACACGTACTGCGGATTATCGCGCT-5’

Phases of PCR:


Annealing (45 – 65 °C)



Final Elongation

Cycle

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5’-CATGTGCATGACGCCTAATAGCGCGA-3’ I I I I I I I I I I I I I I 3’-GTACACGTACTGCGGATTATCGCGCT-5’

5’-GTGCATG-3’

3’-TTATCGC-5’

5’-CATGTGCATGACGCCTAATAGCGCGA-3’ 3’-GTACACGTACTGCGGATTATCGCGCT-5’

Primer 2 5’-GTGCATG-3’

Primer 1 3’-TTATCGC-5’

Taq Pol.

Phases of PCR:



Elongation (72°C)


Final Elongation

Cycle

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5’-CATGTGCATGACGCCTAATAGCGCGA-3’ I I I I I I I I I I I I I I 3’-GTACACGTACTGCGGATTATCGCGCT-5’

5’-GTGCATG-3’

3’-TTATCGC-5’ Taq Pol.

Taq Pol.

5’-CATGTGCATGACGCCTAATAGCGCGA-3’ I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 3’-GTACACGTACTGCGGATTATCGCGCT-5’

5’-GTGCATGACGCCTAATAGCG-3’

3’-CACGTACTGCGGATTATCGC-5’

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History of PCR

1971 1976

1971:

• Norwegian postoc Kjell Kleppe (in Khorana’s lab) envisioned a process very similar to PCR

• Article in the Journal of Molecular Biology first described a method using an enzymatic assay to replicate a short DNA template with oligonucleotides in vitro

• Early form of PCR did not receive much attention due to • Difficulty and costs of oligonucleotide production • No thermostable DNA polymerase • No automated thermocycling instruments

1983 1986 1993 1976

1976:

• Isolation of thermostable (above 75°C) DNA polymerase from Thermus aquaticus from hot springs

https://microbewiki.kenyon.edu/index.php/ Thermus_aquaticus* Image: Flickr/Don Graham). blogs.discovermagazine.com

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History of PCR

1971 1976

1983:

• Invention of the PCR is credited to Kary Mullis, a researcher at Cetus Corporation (one of the first biotechnology companies)

• Water bath PCR (either 3 different water baths or one constantly heating and cooling water bath system)

• DNA Polymerase has to be replaced after each thermal cycle

1983 1986 1993

1986:

• Use of thermostable Taq DNA polymerase enables development of modern thermocyclers

http://www.edvotek.com/544_gallery.png

http://rachenmay.angelfire.com/mullis.jpg

http://en.wikipedia.org/wiki/Thermal_cycler

1986 1993

1993:

• Kary Mullis wins Nobel prize for the invention of PCR

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Advancement of PCR

1992

Conventional PCR

• Endpoint detection via agarose gel electrophoresis

• Limited quantification possible in a small time window

Now

http://en.wikipedia.org/wiki/Thermal_cycler

Log

(co

pie

s)

Cycle

1

2

3

a b c

a b c

1 2 3

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Advancement of PCR

1992

1992:

• Higuchi et al. demonstrated the simultaneous amplification and detection of DNA by adding the intercalating dye ethidium bromide (EtBr)

• Detection of resulting fluorescence after irradiation with UV light

• Fluorescence accumulation is related to the number of starting DNA copies. Fewer cycles are needed to produce a detectable signal when a greater number of target molecules are present

=> First “real-time” system

Now

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Advancement of PCR

1992

Real-time PCR – quantitative PCR

• Direct simultaneous amplification and detection of nucleic acids

• Quantification of DNA amount via fluorescence signal

• Increasing amount of PCR product creates increasing fluorescence

• Non-specific fluorescent dyes (EtBr, SYBRGreen, ...)

• Sequence-specific fluorescent probes (TaqMan probes, Molecular Beacon, ...)

Now

Flu

ore

sce

nce

Cycle

Threshold cT1 cT2

Amplification of minimal amounts of DNA for subsequent analysis, such as cloning,

sequencing, visualisation Applications:

• Genetic testing - detect mutations • Forensics - genetic fingerprinting • Detecting oncogene mutations in cancer • Phylogenetic analysis - building phylogenetic trees • Quantification of virus load • Diagnostics in infectious disease – direct detection of pathogens

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Application of PCR

1992 Now

PCR in Lyme diagnostics

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1989:

• First PCR assay for specific detection of B. burgdorferi s.l. was reported:

Rosa, P. A., and T. G. Schwan. 1989. A specific and sensitive assay for the Lyme disease spirochete Borrelia burgdorferi using the polymerase chain reaction. J. Infect. Dis. 160:1018–1029.

Up to now:

• >5000 publications on PCR detection of Borrelia

• Mostly used for research purposes

• Less clinical application for diagnostics

Literature on PCR in Lyme diagnostics

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Cerar et al., 2008: • 11.9% of blood samples and 15.4% of CSF of patients with features of neuroborreliosis

were positive in PCR

Moniuszko et al., 2015: • 2% of whole blood and 48% of skin biopsy samples were PCR positive in EM patients

from Poland

Lebech et al., 2001: • 71% of skin biopsy and 13% of urine samples of patients with EM and 17% of CSF and

7% of urine samples of patients with neuroborreliosis were positive in PCR

Priem et al., 1997: • Diagnostic sensitivity of PCR in patients with Lyme arthritis: 85% with SF samples, 79% with urine samples, and 91% with paired SF-urine samples

• Sensitivity of PCR in patients with neuroborreliosis: 79% with CSF samples, 45% with urine samples, and 87% with paired CSF-urine

specimens

Challenges of PCR in Lyme diagnostics

Specificity: 98-100% due to specific primer sequences.

Sensitivity: • Analytical sensitivity relatively easy to determine in experiments

• Overall detection sensitivity for individual patient varies with bacterial load depending on:

stage of disease of the patient (acute, chronic, re-activation)

the type of material (blood, serum, CSF, skin biopsy, synovial fluid or tissue)

the quality and the volume deployed for PCR (fresh, frozen, …)

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Appropriate procedures for sample collection, transport and DNA extraction are critical for reliable results

Sensitivities for detection of B. burgdorferi s.l. using PCR

Reviewed by

Aguero-Rosenfeld et al., 2005

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Clinical specimen

region

Range of

sensitivity

[%]

Median

sensitivity

No. of

studies

included

Skin biopsy

EM 36-88 69% 16

United States 59-67 64% 4

Europe 36-88 73% 12

ACA (Europe) 54-100 76% 8

Blood, plasma, serum 0-100 14% 6


Europe 4-100 10% 3

CSF 12-100 38% 16


Europe 12-100 23% 10

Synovial fluid 42-100 78% 8


Europe 42-85 66% 4

Clinical specimen

region

Range of

sensitivity

[%]

Median

sensitivity

No. of

studies

included

Skin biopsy

EM 75-80 78% 3

CSF 15-30 28% 3

Synovial fluid 60-85 73% 2

Borchers et al., 2014

Development of in-house Borrelia-PCR in BCA-lab

Aim: Improve diagnostics of tick-borne disease (Detection of Borrelia miyamotoi included)

Collect in-house knowledge and expertise

Enable Borrelia species identification by direct sequencing of PCR products

Set-up a Borrelia DNA collection for epidemiological and phylogenetic analysis

Increase Sensitivity

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1

2

1. Round of PCR amplification using outer primers

2. Round of PCR amplification using inner primers

Outer primers Inner primers

BCA Approach:

• Combination of several PCR reactions targeting at least two (often three) different genes (16S rDNA, flaB, IGS)

• nested PCR

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Stand:201601

DNS Extraktion aus 10 ml EDTA-Blut SAAF201

(10 ml EDTA Blut Reserve 4°C, anschl. -80°C)

Fließschema Detektion von Borrelia (inkl. B. miyamotoi)- Routine Laborauftrag

Borrelia burgdorferi-PCR aus Blut (20 ml)

16S nested-PCR Nachweis von Spirochäten (Borrelia)

DNS, inkl. B. miyamotoi

Borrelien-Nachweis negativ

„Es konnte keine Borrelien-DNS

nachgewiesen werden.“

IGS nested-PCR Nachweis allerBorrelia Spezies DNS inkl.

B. miyamotoi

flaB nested-PCR Nachweis von Borrelia burgdorferi s.l.

DNS

Borrelia Nachweis grenzwertig

„Es konnte Spirochäten-DNS nachgewiesen werden, allerdings entspricht die DNS

keinem bisher bekannten Erreger“ => Rücksprache

mit V. Krey

Borrelien-Nachweis mittels in-house PCR

positiv

„Es konnte Borrelien-DNS nachgewiesen

werden.“

16S und IGS PCR positiv

negativ

16S und flaB PCR negativ

16S positiv flaB negativ

16S und flaB PCR positiv

16S negativ flaB positiv

16S positiv IGS negativ

Wiederholung der 16S nested-PCR

2x 16S positiv flaB negative IGS negativ

Borrelien-Nachweis negativ

BCA In-house PCR

+

Borrelia-PCR Specificity

Specificity: 100%, due to specific primer sequences.

PCR specificity was determined according to

• experimental assays • in-silico BLAST analysis performed in BCA research lab

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5’-CATGTGCATGACGCCTAATAGCGCGA-3’ I I I I I I I 3’-TTATCGC-5’


Specificity of flaB nested-PCR

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Bb Ba Bg Bbav Bs Bv Bh Td Barth Bartq

Bb: B. burgdorferi B31 DSM 4680 Ba: B. afzelii DSM10508 Bg: B. garinii DSM10534 Bbav: B. bavariensis DSM23469 Bs: B. spielmanii DSM16813 Bv: B. valaisiana DSM21467 Bh: B. hermsii DSM5251 Td: Treponema denticola DSM 14222 Barth: Bartonella henselae Bartq: Bartonella quintana

389 bp

Specificity of 16S nested-PCR

Bb Ba Bg Bbav Bs Bv Bh Td Barth Bartq

244 bp


List of Borrelia spezies detected with BCA Borrelia-PCR

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B. afzelii B. bavariensis B. burgdorferi sensu stricto B. garinii B. lusitaniae B. spielmanii B. valaisiana B. miyamotoi B. anserina B. bissettii B. carolinensis

B. chilensis B. coriaceae B. crocidurae B. duttonii B. hermsii B. japonica B. parkeri B. persica B. recurrentis B. turcica B. turicatae

12/2015

detects all known causative agents of Lyme disease constantly updated to include new species as soon as the sequences are accessible in the peer-reviewed database.

Challenge of PCR in Lyme diagnostics

Sensitivity!

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BCA-lab’s strategy

Use increased blood volume (min. 10 ml)

Use nested PCR reaction => enrichment of PCR product

Use specific pre-analytic procedure to reduce human host DNA background and enrich bacterial DNA

Pre-analytic procedure

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MolYsisTM Complete, Molzym

Analytical sensitivity

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Sensitivity of 16S nested-PCR

480 bp

244 bp

106 1

B. burgdorferi B31 (DSM4680) genome copies

105 104 103 102 10 107

B. burgdorferi B31 (DSM4680) DNA

50 25 12.5 6.25

Analytical sensitivity

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Sensitivity of flaB nested-PCR

497 bp 389 bp

106 1

B. burgdorferi B31 (DSM4680) genome copies

105 104 103 102 107

B. burgdorferi B31 (DSM4680) DNA

50 25 12.5 6.25

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Starting from Ticks

Patient’s blood Patient’s tissue (skin biopsy, cystic tissue, joint tissue)

Body fluids (synovial fluid, urine)

www.eltern.de www.ihdsl.de

Application of Borrelia-PCR

Application of Borrelia-PCR - results

• Tick project:

Screening of 177 ticks actively collected from golf courses:

9% of all ticks tested positive in Borrelia-PCR

• Patient ticks: 13 of 30 ticks (43%) sent by patients were positive in Borrelia-PCR, ticks sucked on human host Identification of B. burgdorferi s.s., B. afzelii, B. valaisiana

• Patient blood: 2 of 17 blood samples (12%) positive in Borrelia-PCR from patients with recent tick bite and EM

• Skin biopsy of EM region

• Synovial liquid, synovial tissue

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Application of PCR for Co-infections

02/2016:

Bartonella-PCR in place

• Commercial kit using real-time PCR technique

• Same pre-analytic procedure as for Borrelia

During 2016:

Development and establishment of PCR detection assays are planned for

• Babesia

• Anaplasma

• Chlamydia

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BCA PCR - Benefits

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Improve diagnostics of tick-borne disease in BCA-clinic

Highly Specific

• Using specific primers

• Targeting different pathogen genes (≥2)

Highly Sensitive

• Optimised extraction procedure

• Increased extraction volume, pathogen enrichment

• Using sensitive PCR technique

≤ 25 Borrelia genome copies

Fast & reliable

• Results within 2 weeks

• In-house expertise and constant updating

Thank you for your attention!

BCA-research:

Dr. Viktoria Krey

Tel. +49 (0) 821 – 455 471 - 22

e-mail: [email protected]

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mailto:[email protected]



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Fighting the bad bugs!

Questions?

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