A multiplex PCR assay for the simultaneous detection and discrimination of the seven Eimeria species that infect domestic fowl

被引:79
作者
Fernandez, S [1 ]
Pagotto, AH [1 ]
Furtado, MM [1 ]
Katsuyama, AM [1 ]
Madeira, AMBN [1 ]
Gruber, A [1 ]
机构
[1] USP, Dept Pathol, Fac Med Vet & Zootecn, BR-05508000 Sao Paulo, Brazil
关键词
Eimeria; domestic fowl; multiplex; polymerase chain reaction; Sequence-Characterised Amplified Region (SCAR); molecular diagnosis; AMPLIFIED POLYMORPHIC DNA; ARBITRARY PRIMERS; CHICKEN EIMERIA; TENELLA; IDENTIFICATION; DIAGNOSIS; MARKERS; PARASITES; DIFFERENTIATION; VARIABILITY;
D O I
10.1017/S0031182003003883
中图分类号
R38 [医学寄生虫学]; Q [生物科学];
学科分类号
07 ; 0710 ; 09 ; 100103 ;
摘要
This study reports the development of a novel multiplex PCR assay based on SCAR (Sequence-Characterised Amplified Region) markers for the simultaneous diagnosis of the 7 Eimeria species that infect domestic fowl. Primer pairs specific for each species were designed in order to generate a ladder of amplification products ranging from 200 to 811 bp. Sensitivity tests for each species were carried out, showing a detection threshold of 1-5 pg, which corresponds approximately to 2-8 sporulated oocysts. Distinct isolates of the 7 Eimeria species from different geographical sources were tested and successfully detected by the assay. All the species were amplified homogeneously, whether or not one of them was present in a high quantity, indicating that there was no cross-interference. The assay was also tested with different sources of Taq DNA polymerase and thermocycler models, confirming the high reproducibility of the reaction. The economy of consumables and labour represented by a single-tube reaction greatly facilitates the molecular diagnosis of a large number of samples, making it appropriate for field epizootiological surveys. We propose the use of this multiplex PCR assay as a rapid and cost-effective diagnostic method for the detection and discrimination of the 7 Eimeria species that infect domestic fowl.
引用
收藏
页码:317 / 325
页数:9
相关论文
共 39 条
[31]   INTRA-SPECIFIC VARIATION WITHIN EIMERIA-TENELLA DETECTED BY THE RANDOM AMPLIFICATION OF POLYMORPHIC DNA [J].
SHIRLEY, MW ;
BUMSTEAD, N .
PARASITOLOGY RESEARCH, 1994, 80 (04) :346-351
[32]   ENZYME VARIATION IN EIMERIA SPECIES OF CHICKEN [J].
SHIRLEY, MW .
PARASITOLOGY, 1975, 71 (DEC) :369-&
[33]  
Shirley MW, 1995, BIOTECHNOLOGY GUIDEL, P1
[34]   EIMERIA-TENELLA - CHARACTERIZATION OF A 5S RIBOSOMAL-RNA REPEAT UNIT AND ITS USE AS A SPECIES-SPECIFIC PROBE [J].
STUCKI, U ;
BRAUN, R ;
RODITI, I .
EXPERIMENTAL PARASITOLOGY, 1993, 76 (01) :68-75
[35]   Discrimination of eight chicken Eimeria species using the two-step polymerase chain reaction [J].
Tsuji, N ;
Kawazu, S ;
Ohta, M ;
Kamio, T ;
Isobe, T ;
Shimura, K ;
Fujisaki, K .
JOURNAL OF PARASITOLOGY, 1997, 83 (05) :966-970
[36]   FINGERPRINTING GENOMES USING PCR WITH ARBITRARY PRIMERS [J].
WELSH, J ;
MCCLELLAND, M .
NUCLEIC ACIDS RESEARCH, 1990, 18 (24) :7213-7218
[37]   DNA POLYMORPHISMS AMPLIFIED BY ARBITRARY PRIMERS ARE USEFUL AS GENETIC-MARKERS [J].
WILLIAMS, JGK ;
KUBELIK, AR ;
LIVAK, KJ ;
RAFALSKI, JA ;
TINGEY, SV .
NUCLEIC ACIDS RESEARCH, 1990, 18 (22) :6531-6535
[38]   Single-strand restriction fragment length polymorphism analysis of the second internal transcribed spacer (ribosomal DNA) for six species of Eimeria from chickens in Australia [J].
Woods, WG ;
Whithear, KG ;
Richards, DG ;
Anderson, GR ;
Jorgensen, WK ;
Gasser, RB .
INTERNATIONAL JOURNAL FOR PARASITOLOGY, 2000, 30 (09) :1019-1023
[39]   A simple method of DNA extraction for Eimeria species [J].
Zhao, XM ;
Duszynski, DW ;
Loker, ES .
JOURNAL OF MICROBIOLOGICAL METHODS, 2001, 44 (02) :131-137