Identificarea / discriminarea Identificarea / discriminarea tulpinilor de tulpinilor de TrichinellaTrichinella spp. prin spp. prin

multiplex PCR clasicmultiplex PCR clasic

Dr. Virgilia POPADr. Virgilia POPA , Prof. Dr. Dumitru MILITARU , Prof. Dr. Dumitru MILITARU INSTITUTUL PASTEUR INSTITUTUL PASTEUR

SimpozionulSimpozionul REALIZARI, PRIORITATI SI PERSPECTIVE IN DOMENIUL REALIZARI, PRIORITATI SI PERSPECTIVE IN DOMENIUL

MEDICINEI VETERINAREMEDICINEI VETERINARE

Academia de Stiinte Agricole si Silvice “Gheorghe Ionescu – Sisesti”Academia de Stiinte Agricole si Silvice “Gheorghe Ionescu – Sisesti”Sectia de Medicina VeterinaraSectia de Medicina Veterinara

Bucuresti 15.06.2012Bucuresti 15.06.2012

Nr. crt.

Specie Genotip Grup Gazda Distributie

1

T. spiralis T1 Incapsulata Mamifere (suine domestice / salbatice, animale sinantropice, carnivore salbatice)

Globala

2T. nativa T2 Incapsulata Mamifere (carnivore

salbatice)Zone arctice / subarctice - Europa, America, Asia

3T6 Zone arctice / subarctice - America

4T. britovi T3 Incapsulata Mamifere (carnivore

silvatice)Zone temperate - Europa, Asia; nordul si vestul Africii

5 T8 Africa de Sud, Namibia

6 T. pseudospiralis T4 Non-incapsulata Mamifere si pasari Globala

7 T.murelli T5 Incapsulata Mamifere Zone temperate - America de Nord

8 T9 Japonia

9 T. nelsoni T7 Incapsulata Mamifere Estul si sudul Africii

10 T. papuae T10 Non-incapsulata Mamifere si reptile Papua Noua Guinee

11 T. zimbabwensis T11 Non-incapsulata Mamifere si reptile Africa Sub-Sahariana

Trichineloza este o parazitoza zoonotica de origine Trichineloza este o parazitoza zoonotica de origine alimentara, datorata unui helmint / nematod din alimentara, datorata unui helmint / nematod din familia familia TrichinellidaeTrichinellidae, al carei prim reprezentant a , al carei prim reprezentant a fost descris in 1835.fost descris in 1835.

T. spiralisT. spiralis,, preponderent preponderent porc domestic, porc porc domestic, porc

salbatic salbatic

T. britoviT. britovi, animale , animale salbatice (urs brun, lup, salbatice (urs brun, lup, mistret, pisica salbatica mistret, pisica salbatica

etc).etc).

International Trichinella Reference CenterInternational Trichinella Reference Center / /

European Reference Laboratory for Parasites (EURLP)European Reference Laboratory for Parasites (EURLP)

http://www.iss.it/site/Trichinella/scripts/sear.asphttp://www.iss.it/site/Trichinella/scripts/sear.asp

LNR pentru LNR pentru TrichinellaTrichinella de la IISPV, de la IISPV,

Colectivul Prof. Dr. V. Cozma, FMV ClujColectivul Prof. Dr. V. Cozma, FMV Cluj

Dr. IV Patrascu, Serviciile veterinareDr. IV Patrascu, Serviciile veterinare

RomaniaRomania1999 – 20111999 – 2011

Commission Regulation (EC) No 2075/2005 of 5 December 2005 laying down specific rules on Commission Regulation (EC) No 2075/2005 of 5 December 2005 laying down specific rules on official controls for official controls for TrichinellaTrichinella in meat (amended by Ec1665/2006, Ec1245/2007, Ec1109/2011) in meat (amended by Ec1665/2006, Ec1245/2007, Ec1109/2011)

EURLP: Guidelines for the validation of apparatuses for the EURLP: Guidelines for the validation of apparatuses for the detection of detection of TrichinellaTrichinella larvae, 2008 (DG Sanco approved) larvae, 2008 (DG Sanco approved)

EURLP: Guideline for the detection of EURLP: Guideline for the detection of TrichinellaTrichinella larvae at the larvae at the slaughterhouse or connected laboratory in a Quality slaughterhouse or connected laboratory in a Quality Assurance System, 2006Assurance System, 2006

EURLP: Guidelines for the identification and development of EURLP: Guidelines for the identification and development of sampling methods and design of suitable protocols for sampling methods and design of suitable protocols for monitoring of monitoring of TrichinellaTrichinella infection in indicator species, 2004 infection in indicator species, 2004

Manual OIE: Manual OIE: C h a p t e r 2 . 1 . 1 6 . TrichinellosisC h a p t e r 2 . 1 . 1 6 . Trichinellosis Manual OIE: C h a p t e r 1 . 1 . 3 . Quality management in veterinary testing laboratoriesManual OIE: C h a p t e r 1 . 1 . 3 . Quality management in veterinary testing laboratories Manual OIE: C h a p t e r 1 . 1 . 4 / 5 . Principles and methods of validation of diagnostic assays for infectious diseasesManual OIE: C h a p t e r 1 . 1 . 4 / 5 . Principles and methods of validation of diagnostic assays for infectious diseases ISO17025:2005ISO17025:2005 EA-04/10:2002, rev.02, Accreditation for Microbiological LaboratoriesEA-04/10:2002, rev.02, Accreditation for Microbiological Laboratories

Identificarea de specie este necesara pentru un Identificarea de specie este necesara pentru un diagnostic corect, pe baza caruia sa poata fi stabilite diagnostic corect, pe baza caruia sa poata fi stabilite coordonatele epidemiologice si luate masuri sanitare coordonatele epidemiologice si luate masuri sanitare veterinare. veterinare.

Mai mult, aceasta identificare trebuie realizata la nivel Mai mult, aceasta identificare trebuie realizata la nivel de de 1 larva (1MSL)1 larva (1MSL), deoarece au fost diagnosticate , deoarece au fost diagnosticate cazuri cu infectii in care erau prezente concomitent, cazuri cu infectii in care erau prezente concomitent, pe acelasi animal, cel putin 2 specii de pe acelasi animal, cel putin 2 specii de TrichinellaTrichinella

2009, porc domestic, jud. Galati: EURLP a confirmat 2009, porc domestic, jud. Galati: EURLP a confirmat infectia mixtainfectia mixta cu cu T. spiralisT. spiralis si si T. britoviT. britovi

Identificarea de specie la Identificarea de specie la TrichinellaTrichinella poate fi realizata prin poate fi realizata prin

metode fenotipice, parazitologice (consumatoare de timp si care necesita un grad deosebit de inalt de expertiza parazitologica)metode fenotipice, parazitologice (consumatoare de timp si care necesita un grad deosebit de inalt de expertiza parazitologica)

metode genetice (moleculare, care sunt mult mai rapide si care necesita experienta de biologie moleculara)metode genetice (moleculare, care sunt mult mai rapide si care necesita experienta de biologie moleculara)

-amplificare clasica – amplificare clasica – control post-amplificare control post-amplificare prin gel-electroforeza prin gel-electroforeza (end-point)(end-point) - amplificare in timp real - amplificare in timp real (spectrofotometrica) – (spectrofotometrica) – control in timpul reactiei control in timpul reactiei

Identificarea Identificarea TrichinellaTrichinella spp. spp.Autor Articol Tip PCR Secventa (e) Discriminare LOD

Zarlenga et al. 1999 A multiplex PCR for unequivocal differenciation of all encapslated and non-encapsulated genotypes of Trichinella, Int.J.Paras. 29:1858-67

mPCR clasic (multi-amorsa / multi-primer)

ITS1, ITS2 (internal transcribed spacers) si ESV (expansion segment V), din regiunea repetitiva a ADN-ului ribosomal

9 genotipuri / 8 specii Trichinella

> 5 - 10 ng DNA

Blaga et al. 2009 Use of mitochondrial RNA genes for thedifferentiation of four Trichinella species bymultiplex PCR amplification, Journal of Helminthology 83: 121–128

mPCR clasic (multi-amorsa / multi-primer)

mt-rrnS, mt-rrnL 4 specii Trichinella 100 pg DNA

Popa et al. 2009 Molecular studies regarding to the differentiation between Trichinella spiralis and T. pseudospiralis, Lucrări Stiinţifice de Medicină Veterinară USAMV Timişoara 42 (1): 116-124, ISSN:1221-5295)

rPCR / rTq-PCR

hsp70 / shsp 2 specii Trichinella < 5 pg DNA

Identificarea Identificarea TrichinellaTrichinella spp. spp.

Larve de referinta Larve de referinta EURLP / IPEURLP / IP

T. spiralisT. spiralis (ISS3 / IP) (ISS3 / IP) T.pseudospiralisT.pseudospiralis (ISS) (ISS) T. britoviT. britovi (ISS2) (ISS2) T. nativaT. nativa (ISS10) (ISS10)

Identificarea Identificarea TrichinellaTrichinella spp. spp.Extractia ADN – 1 MSL

Liza termica si Liza termica si enzimatica (Sigma)enzimatica (Sigma)

QiaAmp DNA mini kit QiaAmp DNA mini kit (Qiagen)(Qiagen)

Tehnologia Tehnologia paramagnetica paramagnetica (Promega (Promega DC6740DC6740 / / DC6701)DC6701)

Identificarea Identificarea TrichinellaTrichinella spp. spp.Extractia ADN – 1 MSL Cuantificare ADN

Qubit (Invitrogen) – fluorimetrie – Qubit (Invitrogen) – fluorimetrie – LOD >5pg/ulLOD >5pg/ul

HPLC (Waters) – RP si perechi de HPLC (Waters) – RP si perechi de ioni – LOD ioni – LOD >>500 pg/ul500 pg/ul

AU

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

Identificarea Identificarea TrichinellaTrichinella spp. spp.Extractia ADN – 1 MSL

Cuantificare ADN prin rPCR – UCO

Identificarea Identificarea TrichinellaTrichinella spp. spp.Cuantificare ADN prin rPCR – UCO Anova P = 0.1981

Concentratie ADN (numar copii)

Sure + Mx

Sure + PE48

Sure+ iCy48

Maxima + Mx

Maxima + PE48

Maxima + iCy48

Maxima + iCy5T

Tb1/2-02-12 20.76 8.65E+06 2.16E+07 6 11 7 9Tb1/31-01-12 20.85 8.04E+06 2.01E+07 6Tb2/2-02-12 20.04 1.55E+07 3.88E+07 6 15 16, 20 12Tb2/31-01-12 20.78 8.55E+06 2.14E+07 6Tb-ISS 21.03 20.39 1.17E+07 2.93E+07 36 28, 31, 33

Media 20.56 1.05E+07 2.62E+07Deviatia standard 0.34 3.14E+06 7.88E+06

Tn1/2-02-12 20.42 1.14E+07 2.85E+07 6 11 7 9Tn2/2-02-12 20.71 9.00E+06 2.25E+07 6 15 16, 20 12Tn-ISS 21.03 18.71 4.50E+07 1.13E+08 36 28, 31, 33

Media 19.95 2.18E+07 5.45E+07Deviatia standard 1.08 2.02E+07 5.03E+07

Tps2-ISS 21.03 20.8 8.40E+06 2.10E+07 36 28, 31, 33Tps1-ISS 29.11 22.03 7.35E+06 1.84E+07 150, 173 151 152

Media 21.42 7.88E+06 1.97E+07Deviatia standard 0.87 7.42E+05 1.86E+06

Ts1/2-02-12 20.5 1.07E+07 2.68E+07 6 11 7 9Ts2/2-02-12 20.86 7.99E+06 2.00E+07 6 15 16, 20 12Ts-IP 21.03 25.3 2.26E+05 5.65E+05 36 28Ts-ISS 21.03 23.46 9.93E+05 2.48E+06 36 28, 31, 33

Media 22.53 4.97E+06 1.24E+07Deviatia standard 2.27 5.16E+06 1.29E+07

Tspp-Obor 29.11 21.29 5.64E+06 1.41E+07 150, 36 151 20Tspp-Timis 28.02 21.46 4.93E+06 1.23E+07 36 21Tspp-Tulcea 14.03 23.96 6.67E+05 1.67E+06 36 27

Media 22.24 3.75E+06 9.36E+06Deviatia standard 1.49 2.69E+06 6.72E+06

UCO-Sybr 10000copii 28.77 1.00E+04UCO-Sybr 1000copii 31.45 1.00E+03UCO-Sybr 100copii 36.21 1.00E+02UCO-Sybr 10copii 38.73 1.00E+01UCO-Sybr 1copie 39.48 1.00E+00

FLLarva MS Ct

Volum

4ul 10ul

Identificarea Identificarea TrichinellaTrichinella spp. spp.Enzime / amestecuri / programe / Enzime / amestecuri / programe /

aparate de amplificareaparate de amplificare

Tsp

p /

Pro

gram

/

Apa

rat

Dat

a ex

trac

tiei

Sur

e

Max

Taq

Dat

a ex

trac

tiei

Sur

e

Max

Taq

Dat

a ex

trac

tiei

Sur

e

Max

Taq

Dat

a ex

trac

tiei

Sur

e

Max

Taq

Dat

a ex

trac

tiei

Sur

e

Max

Taq

Ts (IP) 29.11 12.12 (p20)

PE48 151 148

iCy48 159 157 (-)

iCy5T 156 (-) 172; 5 162; 165;

161;

Mx 150 173; 6

TS-ISS3 31.01. 2.02

PE48 15 7; 16 8 (±)

iCy48 9 10 (-)

iCy5T 5 12 13 (-)

Mx 6 11 ((±)

Tps-1 29.11 12.12 (p20)

PE48 151 148 7; 16 8

iCy48 152

iCy5T 156 (±); 160 (-)

172; 5 162; 165;

161;

Mx 150 173; 6

Tps2 7.12 12.12 (p20)

PE48

iCy48 159 157 (-) 9 10

iCy5T 172 162; 165; 12

161; 13 (-)

Mx 173 11

Tb-ISS2 31.01 2.02

PE48 15 7; 16 8 (±)

iCy48 9 10 (-)

iCy5T 5 (-) 12 13 (-)

Mx 6 6 11 ((±)

Tn-ISS10 31.01 2.02

PE48 15 7; 16 8 (±)

iCy48 9 10 (-)

iCy5T 5 12 13 (-)

Mx 6 11 (±)

Identificarea Identificarea TrichinellaTrichinella spp. spp.

Multiplex PCR pentru identificarea Trichinella spp. Gel-electroforeza agaroza 2.5%, TBE1x. A. Maxima SYBR Green/ROX qPCR Master Mix 2X (Fermentas K0221). Liniiile 1-5: PE48 Liniile 7-10: iCy48. Liniile 12-15: iCy5T. Linia 1: NFW; liniile 2, 7, 12: T. spiralis ISS; liniile 3, 8, 13: T. britovi ISS; liniile 4, 9, 14: T. nativa ISS; liniile 5, 10, 15: T.pseudospiralis ISS; linia 6: 100 bp DNA Ladder (Promega); linia 11: 50 bp Step Ladder (Sigma). B: 2x Taq PCR Master Mix (Promega M7505). Liniiile 1-5: PE48 Liniile 7-10: iCy48. Liniile 12-15: iCy5T. Linia 1: NFW; liniile 2, 7, 12: T. spiralis ISS; liniile 3, 8, 13: T. britovi ISS; liniile 4, 9, 14: T. nativa ISS; liniile 5, 10, 15: T.pseudospiralis ISS; linia 6: 100 bp DNA Ladder (Promega); linia 11: 50 bp Step Ladder (Sigma).

Multiplex PCR pentru identificarea Trichinella spp. Gel-electroforeza agaroza 2.5%, TBE1x. 2x Brilliant II Sybr Green Q-PCR Master Mix, Agilent / Stratagene 600828. A: Mx3005P, program 29. Linia 1: NFW; liniile 2,3, 9: T.spiralis (IP); liniile 4,5, 8: T.pseudospiralis ISS, lot1. B: PE48. Linia 1: NFW; liniile 2,3, 9: T.spiralis (IP); liniile 4,5, 8: T.pseudospiralis ISS, lot1.

Enzime / amestecuri / programe / Enzime / amestecuri / programe / aparate de amplificareaparate de amplificare

ConcluziiConcluzii Metoda de identificare / discriminare a speciilor / genotipurilor de Metoda de identificare / discriminare a speciilor / genotipurilor de TrichinellaTrichinella prin amplificare PCR de tip multiamorsa cf. procedurii prin amplificare PCR de tip multiamorsa cf. procedurii EURLP adaptata prin procedura interna a fost validata in conditiile EURLP adaptata prin procedura interna a fost validata in conditiile Laboratorului de biologie moleculara din I.Pasteur, prin specificitate Laboratorului de biologie moleculara din I.Pasteur, prin specificitate si repetabilitate, pe materialele de referinta EURLP.si repetabilitate, pe materialele de referinta EURLP.

Enzimele, respectiv amestecurile de amplificare, care s-au validat Enzimele, respectiv amestecurile de amplificare, care s-au validat sunt Maxima SYBR Green/ROX qPCR Master Mix 2X (Fermentas sunt Maxima SYBR Green/ROX qPCR Master Mix 2X (Fermentas K0221) si Brilliant II Sybr Green Q-PCR Master Mix (Agilent / K0221) si Brilliant II Sybr Green Q-PCR Master Mix (Agilent / Stratagene 600828)Stratagene 600828)

Concentratia de ADN obtinut dintr-o singura larva s-a situat sub 0.5 Concentratia de ADN obtinut dintr-o singura larva s-a situat sub 0.5 pg/ul, indiferent de specia sau genotipul de pg/ul, indiferent de specia sau genotipul de TrichinellaTrichinella..

Prin extractia paramagnetica a ADN, realizata in laboratorul de Prin extractia paramagnetica a ADN, realizata in laboratorul de biologie moleculara din IP, si controlata prin spectrofotometrie cu biologie moleculara din IP, si controlata prin spectrofotometrie cu amplificare enzimatica (qPCR), nu s-au inregistrat diferente datorate amplificare enzimatica (qPCR), nu s-au inregistrat diferente datorate matricei initiale definite de specia / genotipul de matricei initiale definite de specia / genotipul de TrichinellaTrichinella..

ConcluziiConcluzii

Metoda de identificare / discriminare a speciilor / Metoda de identificare / discriminare a speciilor / genotipurilor de genotipurilor de TrichinellaTrichinella prin amplificare PCR de tip prin amplificare PCR de tip multiamorsa este in curs de acreditare ISO17025.multiamorsa este in curs de acreditare ISO17025.

Metoda de identificare / discriminare a speciilor / Metoda de identificare / discriminare a speciilor / genotipurilor de genotipurilor de TrichinellaTrichinella prin amplificare PCR de tip prin amplificare PCR de tip multiamorsa poate fi inclusa in programele nationale de multiamorsa poate fi inclusa in programele nationale de supraveghere / control privind supraveghere / control privind TrichinellaTrichinella spp. spp.

MultumiriMultumiri Dnei Dr. Rodica Tanasuica, Dnei Dr. Rodica Tanasuica, Director General IISPVDirector General IISPV Dnei Dr. Sofica Apostu, Sef LNR – IISPVDnei Dr. Sofica Apostu, Sef LNR – IISPV Dnei IDr. Tertis Marinela Ofelia,Dnei IDr. Tertis Marinela Ofelia, Director DSVSA TulceaDirector DSVSA Tulcea Dlui Dr. Bria Paul Cornel,Dlui Dr. Bria Paul Cornel, Director tehnic DSVSA TulceaDirector tehnic DSVSA Tulcea Sef de laboratorSef de laborator Dnei Ing. Chim. Victorita Burghelea,Dnei Ing. Chim. Victorita Burghelea, Director General IPasteurDirector General IPasteur Dlui Dr. Niculai Poparlan, Dlui Dr. Niculai Poparlan, Consiler Presedinte SN I.PasteurConsiler Presedinte SN I.Pasteur Dlui Prof. univ. Dr. Valer Teusdea Dlui Prof. univ. Dr. Valer Teusdea Dlui Conf. univ. Dr. Nicolae AlexandruDlui Conf. univ. Dr. Nicolae Alexandru Dnei Ing. Chim. Ana CsumaDnei Ing. Chim. Ana Csuma