This document discusses the identification and differentiation of Trichinella species and genotypes using multiplex PCR. It provides background on Trichinella and trichinellosis. Eleven genotypes of Trichinella are identified that infect various mammal and some reptile hosts. Molecular identification of Trichinella at the single larva level is important for accurate diagnosis and epidemiological investigation. The document describes extraction of DNA from single larvae and quantification methods, then details a multiplex PCR assay that can identify the four main Trichinella species and provide results more rapidly than phenotypic methods.

1. Identificarea / discriminareaIdentificarea / discriminarea
tulpinilor detulpinilor de TrichinellaTrichinella spp. prinspp. prin
multiplex PCR clasicmultiplex PCR clasic
Dr. Virgilia POPADr. Virgilia POPA , Prof. Dr. Dumitru MILITARU, Prof. Dr. Dumitru MILITARU
INSTITUTUL PASTEURINSTITUTUL PASTEUR
SimpozionulSimpozionul
REALIZARI, PRIORITATI SI PERSPECTIVE IN DOMENIULREALIZARI, 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

2. Nr.
crt.
Specie Genotip Grup Gazda Distributie
1
T. spiralis T1 Incapsulata Mamifere (suine
domestice / salbatice,
animale sinantropice,
carnivore salbatice)
Globala
2
T. nativa T2 Incapsulata Mamifere (carnivore
salbatice)
Zone arctice / subarctice - Europa,
America, Asia
3
T6 Zone arctice / subarctice - America
4
T. 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 origineTrichineloza este o parazitoza zoonotica de origine
alimentara, datorata unui helmint / nematod dinalimentara, datorata unui helmint / nematod din
familiafamilia TrichinellidaeTrichinellidae, al carei prim reprezentant a, al carei prim reprezentant a
fost descris in 1835.fost descris in 1835.

3. T. spiralisT. spiralis,, preponderentpreponderent
porc domestic, porcporc domestic, porc
salbaticsalbatic
T. britoviT. britovi, animale, animale
salbatice (urs brun, lup,salbatice (urs brun, lup,
mistret, pisica salbaticamistret, 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 pentruLNR 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
RomaniaRomania
1999 – 20111999 – 2011

4.  Commission Regulation (EC) No 2075/2005 of 5 December 2005 laying down specific rules onCommission Regulation (EC) No 2075/2005 of 5 December 2005 laying down specific rules on
official controls forofficial 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 theEURLP: Guidelines for the validation of apparatuses for the
detection ofdetection of TrichinellaTrichinella larvae, 2008 (DG Sanco approved)larvae, 2008 (DG Sanco approved)
 EURLP: Guideline for the detection ofEURLP: Guideline for the detection of TrichinellaTrichinella larvae at thelarvae at the
slaughterhouse or connected laboratory in a Qualityslaughterhouse or connected laboratory in a Quality
Assurance System, 2006Assurance System, 2006
 EURLP: Guidelines for the identification and development ofEURLP: Guidelines for the identification and development of
sampling methods and design of suitable protocols forsampling methods and design of suitable protocols for
monitoring ofmonitoring of TrichinellaTrichinella infection in indicator species, 2004infection 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

5.  Identificarea de specie este necesara pentru unIdentificarea de specie este necesara pentru un
diagnostic corect, pe baza caruia sa poata fi stabilitediagnostic corect, pe baza caruia sa poata fi stabilite
coordonatele epidemiologice si luate masuri sanitarecoordonatele epidemiologice si luate masuri sanitare
veterinare.veterinare.
 Mai mult, aceasta identificare trebuie realizata la nivelMai mult, aceasta identificare trebuie realizata la nivel
dede 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 depe acelasi animal, cel putin 2 specii de TrichinellaTrichinella
 2009, porc domestic, jud. Galati: EURLP a confirmat2009, porc domestic, jud. Galati: EURLP a confirmat
infectia mixtainfectia mixta cucu T. spiralisT. spiralis sisi T. britoviT. britovi

6. Identificarea de specie laIdentificarea de specie la TrichinellaTrichinella poate fi realizata prinpoate 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)

7. -amplificare clasica –amplificare clasica –
control post-amplificarecontrol post-amplificare
prin gel-electroforezaprin gel-electroforeza
(end-point)(end-point)
- amplificare in timp real- amplificare in timp real
(spectrofotometrica) –(spectrofotometrica) –
control in timpul reactieicontrol in timpul reactiei

8. IdentificareaIdentificarea 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 the
differentiation of four
Trichinella species by
multiplex 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

9. IdentificareaIdentificarea TrichinellaTrichinella spp.spp.
Larve de referintaLarve 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)

10. IdentificareaIdentificarea TrichinellaTrichinella spp.spp.
Extractia ADN – 1 MSL
 Liza termica siLiza termica si
enzimatica (Sigma)enzimatica (Sigma)
 QiaAmp DNA mini kitQiaAmp DNA mini kit
(Qiagen)(Qiagen)
 TehnologiaTehnologia
paramagneticaparamagnetica
(Promega(Promega DC6740DC6740 //
DC6701)DC6701)

11. IdentificareaIdentificarea 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 deHPLC (Waters) – RP si perechi de
ioni – LODioni – 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
Minutes
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

12. IdentificareaIdentificarea TrichinellaTrichinella spp.spp.
Extractia ADN – 1 MSL
Cuantificare ADN prin rPCR – UCO

13. IdentificareaIdentificarea 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 9
Tb1/31-01-12 20.85 8.04E+06 2.01E+07 6
Tb2/2-02-12 20.04 1.55E+07 3.88E+07 6 15 16, 20 12
Tb2/31-01-12 20.78 8.55E+06 2.14E+07 6
Tb-ISS 21.03 20.39 1.17E+07 2.93E+07 36 28, 31, 33
Media 20.56 1.05E+07 2.62E+07
Deviatia standard 0.34 3.14E+06 7.88E+06
Tn1/2-02-12 20.42 1.14E+07 2.85E+07 6 11 7 9
Tn2/2-02-12 20.71 9.00E+06 2.25E+07 6 15 16, 20 12
Tn-ISS 21.03 18.71 4.50E+07 1.13E+08 36 28, 31, 33
Media 19.95 2.18E+07 5.45E+07
Deviatia standard 1.08 2.02E+07 5.03E+07
Tps2-ISS 21.03 20.8 8.40E+06 2.10E+07 36 28, 31, 33
Tps1-ISS 29.11 22.03 7.35E+06 1.84E+07 150, 173 151 152
Media 21.42 7.88E+06 1.97E+07
Deviatia standard 0.87 7.42E+05 1.86E+06
Ts1/2-02-12 20.5 1.07E+07 2.68E+07 6 11 7 9
Ts2/2-02-12 20.86 7.99E+06 2.00E+07 6 15 16, 20 12
Ts-IP 21.03 25.3 2.26E+05 5.65E+05 36 28
Ts-ISS 21.03 23.46 9.93E+05 2.48E+06 36 28, 31, 33
Media 22.53 4.97E+06 1.24E+07
Deviatia standard 2.27 5.16E+06 1.29E+07
Tspp-Obor 29.11 21.29 5.64E+06 1.41E+07 150, 36 151 20
Tspp-Timis 28.02 21.46 4.93E+06 1.23E+07 36 21
Tspp-Tulcea 14.03 23.96 6.67E+05 1.67E+06 36 27
Media 22.24 3.75E+06 9.36E+06
Deviatia standard 1.49 2.69E+06 6.72E+06
UCO-Sybr 10000copii 28.77 1.00E+04
UCO-Sybr 1000copii 31.45 1.00E+03
UCO-Sybr 100copii 36.21 1.00E+02
UCO-Sybr 10copii 38.73 1.00E+01
UCO-Sybr 1copie 39.48 1.00E+00
FL
Larva MS Ct
Volum
4ul 10ul

14. IdentificareaIdentificarea TrichinellaTrichinella spp.spp.
Enzime / amestecuri / programe /Enzime / amestecuri / programe /
aparate de amplificareaparate de amplificare
Tspp/Program/
Aparat
Dataextractiei
Sure
Max
Taq
Dataextractiei
Sure
Max
Taq
Dataextractiei
Sure
Max
Taq
Dataextractiei
Sure
Max
Taq
Dataextractiei
Sure
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 (±)

15. IdentificareaIdentificarea 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

16. ConcluziiConcluzii
 Metoda de identificare / discriminare a speciilor / genotipurilor deMetoda de identificare / discriminare a speciilor / genotipurilor de
TrichinellaTrichinella prin amplificare PCR de tip multiamorsa cf. proceduriiprin amplificare PCR de tip multiamorsa cf. procedurii
EURLP adaptata prin procedura interna a fost validata in conditiileEURLP adaptata prin procedura interna a fost validata in conditiile
Laboratorului de biologie moleculara din I.Pasteur, prin specificitateLaboratorului 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 validatEnzimele, respectiv amestecurile de amplificare, care s-au validat
sunt Maxima SYBR Green/ROX qPCR Master Mix 2X (Fermentassunt 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 0.5Concentratia de ADN obtinut dintr-o singura larva s-a situat sub 0.5
pg/ul, indiferent de specia sau genotipul depg/ul, indiferent de specia sau genotipul de TrichinellaTrichinella..
 Prin extractia paramagnetica a ADN, realizata in laboratorul dePrin extractia paramagnetica a ADN, realizata in laboratorul de
biologie moleculara din IP, si controlata prin spectrofotometrie cubiologie moleculara din IP, si controlata prin spectrofotometrie cu
amplificare enzimatica (qPCR), nu s-au inregistrat diferente datorateamplificare enzimatica (qPCR), nu s-au inregistrat diferente datorate
matricei initiale definite de specia / genotipul dematricei initiale definite de specia / genotipul de TrichinellaTrichinella..

17. ConcluziiConcluzii
 Metoda de identificare / discriminare a speciilor /Metoda de identificare / discriminare a speciilor /
genotipurilor degenotipurilor de TrichinellaTrichinella prin amplificare PCR de tipprin 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 degenotipurilor de TrichinellaTrichinella prin amplificare PCR de tipprin amplificare PCR de tip
multiamorsa poate fi inclusa in programele nationale demultiamorsa poate fi inclusa in programele nationale de
supraveghere / control privindsupraveghere / control privind TrichinellaTrichinella spp.spp.

18. 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 TeusdeaDlui 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