Establishment of ITS-based PCR-RFLP for identifying three species of nematodes in a white yak

Abstract

Abstract:

Objective To develop the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method based on internal transcribed spacers (ITS) of ribosomal DNA to identity three nematode species that infected a white yak. Methods Twelve nematodes were collected from the small intestine of a white yak in Tianzhu Tibetan Autonomous County, Gansu Province, and DNA was extracted. Then the ITS region was amplified by PCR and sequenced. The sequences were analysed by DNAstar7.1 software and the phylogenetic tree was constructed by the neighbor-joining (NJ) method. Specific sites for restriction endonucleases were identified, based on which the PCR products were digested, and the PCR-RFLP method was established. Results PCR resulted in specific ITS bands of 900 bp for all the 12 nematodes. Sequencing results showed that each was approximately 900 bp in length. The sequences of 4 of the 12 nematodes had sequence similarity of > 99% with Cooperia oncophora (GenBank accession number AB534601), Teladorsagia circumcincta (GenBank accession number JF680984), and Ostertagia sp. (GenBank accession number HQ844228). However, the interspecies difference in ITS sequence among the three species was 4.3%-13.9%, while the intraspecific difference was < 1%. The interspecies difference in ITS sequence was most significant between C. Oncophora and T. circumcincta (12.9%-13.1%), medium between C. oncophora and Ostertagia sp. (13.6%-13.9%), and the least between T. circumcincta and Ostertagia sp. (4.3%-4.8%). The phylogenetic tree grouped the three nematode species into three branches, and revealed a closer ralationship between T. circumcincta and Ostertagia sp. PCR-RFLP analyses showed that the ITS sequences of C. Oncophora and T. circumcinctais were cut into fragments of 650 bp and 250 bp with endonuclease NdeⅠ, while only the ITS sequence of C. oncophora was cut into fragments of 700 bp and 200 bp with EcoR Ⅴ. The two endonucleases were ineffective for the ITS sequences of Ostertagia sp. Conclusions The three species of nematodes are successfully identified with ITS-based PCR-RFLP.

Key words: Cooperia oncophora, Teladorsagia circumcincta, Ostertagia sp., ITS, PCR-RFLP

CLC Number:

R383.19

Liang HAN, Dong-hui ZHOU, Qing LIU, Wen-bin ZHEN, Xing-quan ZHU, Xiao-lin SUN. Establishment of ITS-based PCR-RFLP for identifying three species of nematodes in a white yak[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2017, 35(2): 164-168.

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编号Code	TZN1	TZN2	TZN3	TZN4	AB534601	TZN5	TZN6	TZN7	TZN8	JF680984	TZN9	TZN10	TZN11	TZN12	HQ844228
TZN1	-
TZN2	100	-
TZN3	99.9	99.9	-
TZN4	99.9	99.9	100	-
AB534601	99.5	99.5	99.6	99.6	-
TZN5	87.0	87.0	87.1	87.1	86.9	-
TZN6	87.0	87.0	87.1	87.1	86.9	99.7	-
TZN7	86.9	86.9	87.0	87.0	86.8	99.9	99.6	-
TZN8	87.0	87.0	87.1	87.1	86.9	99.7	100	99.6	-
JF680984	86.9	86.9	87.1	87.0	86.8	99.9	99.9	99.7	99.9	-
TZN9	86.3	86.3	86.4	86.4	86.4	95.7	95.7	95.6	95.7	95.6	-
TZN10	86.3	86.3	86.4	86.4	86.4	95.7	95.7	95.6	95.7	95.6	100	-
TZN11	86.1	86.1	86.3	86.3	86.3	95.6	95.6	95.4	95.6	95.4	99.9	99.9	-
TZN12	86.3	86.3	86.4	86.4	86.4	95.7	95.7	95.6	95.7	95.6	100	100	99.9	-
HQ844228	85.8	85.8	85.9	85.9	85.9	95.2	95.2	95.0	95.2	95.0	99.5	99.5	99.6	99.5	-