Objective:To establish a polymerase chain reaction(PCR) technique based on cytochrome b {cytb) gene of mitochondria DNA(mtDNA) for blood meal identification.Methods:The PCR technique was established based on published...Objective:To establish a polymerase chain reaction(PCR) technique based on cytochrome b {cytb) gene of mitochondria DNA(mtDNA) for blood meal identification.Methods:The PCR technique was established based on published information and validated using blood sample of laboratory animals of which their whole gene sequences are available in CenBank.PCR was next performed to compile gene sequences of different species of wild rodents.The primers used were complementary to the conserved region of the cytb gene of vertebrate's mtDNA.A total of 100 blood samples,both from laboratory animals and wild rodents were collected und analyzed.The obtained unknown sequences were compared with those in the GenBank database using BLAST program to identify the vertebrate animal species.Results:Gene sequences of 11 species of wild animals caught in 9 localities of Peninsular Malaysia were compiled using the established PCR. The animals involved were Rattus(rattus) tanezumi,Rattus tiomanicus,Leopoldamys sabanus, Tupaia glis,Tupaia minor,Niviventor cremoriventor,Rhinosciurus laticaudatus,Calloseiurus caniseps,Sundamys muelleri,Rattus rajah,and Maxomys whitelwadi.The BLAST results confirmed the host with exact or nearly exact matches(>89%identity).Ten new gene sequences have been deposited in CenBank database since September 2010.Conclusions:This study indicates that the PCR direct sequencing system using universal primer sets for vertebrate cytb gene is a promising technique for blood meal identification.展开更多
Objective: To identify different stages of Ixodes granulatus(I. granulatus) based on morphological characters prior to molecular identification which is significant for confirming and identifying the nymphal stages of...Objective: To identify different stages of Ixodes granulatus(I. granulatus) based on morphological characters prior to molecular identification which is significant for confirming and identifying the nymphal stages of I. granulatus.Methods: A total of 14 individuals of adult, engorged and nymphal ticks collected from three different localities were examined morphologically using taxonomic keys, followed by PCR using cytochrome oxidase subunit I(COI). Clustering analysis based on COI sequences was carried out by constructing neighbor-joining and maximum parsimony tree to clarify the genetic variation and diversity of local I. granulatus.Results: Based on external morphological characterizations, nine individuals(64.3%)were successfully identified as I. granulatus, while five individuals were recognized only as Ixodes sp. due to lack of morphological characters visible and development during that stage. Molecular analysis of local I. granulatus using COI gene revealed 93%–94% sequence homology from available sequence in Gen Bank and was in concordance with the morphological identification. Furthermore, a low intraspecific variation was observed among the species of I. granulatus collected from different localities(0%–3.7%).Conclusions: These findings demonstrated for the first time the establishment of COI gene for identifying I. granulatus nymphal tick which is of paramount importance to the control of potential tick-borne infections in Malaysia. Moreover, this study provides evidence that a combination of morphology and molecular data was corroborated as an accurate tool for tick identification.展开更多
基金financially supported by a grant(JPP-IMR Code:09-030) from the Ministry of Health,Malaysia
文摘Objective:To establish a polymerase chain reaction(PCR) technique based on cytochrome b {cytb) gene of mitochondria DNA(mtDNA) for blood meal identification.Methods:The PCR technique was established based on published information and validated using blood sample of laboratory animals of which their whole gene sequences are available in CenBank.PCR was next performed to compile gene sequences of different species of wild rodents.The primers used were complementary to the conserved region of the cytb gene of vertebrate's mtDNA.A total of 100 blood samples,both from laboratory animals and wild rodents were collected und analyzed.The obtained unknown sequences were compared with those in the GenBank database using BLAST program to identify the vertebrate animal species.Results:Gene sequences of 11 species of wild animals caught in 9 localities of Peninsular Malaysia were compiled using the established PCR. The animals involved were Rattus(rattus) tanezumi,Rattus tiomanicus,Leopoldamys sabanus, Tupaia glis,Tupaia minor,Niviventor cremoriventor,Rhinosciurus laticaudatus,Calloseiurus caniseps,Sundamys muelleri,Rattus rajah,and Maxomys whitelwadi.The BLAST results confirmed the host with exact or nearly exact matches(>89%identity).Ten new gene sequences have been deposited in CenBank database since September 2010.Conclusions:This study indicates that the PCR direct sequencing system using universal primer sets for vertebrate cytb gene is a promising technique for blood meal identification.
基金Supported by National Institute of Health grant from the Ministry of Health,Malaysia(Code:JPP-IMR 11-010)
文摘Objective: To identify different stages of Ixodes granulatus(I. granulatus) based on morphological characters prior to molecular identification which is significant for confirming and identifying the nymphal stages of I. granulatus.Methods: A total of 14 individuals of adult, engorged and nymphal ticks collected from three different localities were examined morphologically using taxonomic keys, followed by PCR using cytochrome oxidase subunit I(COI). Clustering analysis based on COI sequences was carried out by constructing neighbor-joining and maximum parsimony tree to clarify the genetic variation and diversity of local I. granulatus.Results: Based on external morphological characterizations, nine individuals(64.3%)were successfully identified as I. granulatus, while five individuals were recognized only as Ixodes sp. due to lack of morphological characters visible and development during that stage. Molecular analysis of local I. granulatus using COI gene revealed 93%–94% sequence homology from available sequence in Gen Bank and was in concordance with the morphological identification. Furthermore, a low intraspecific variation was observed among the species of I. granulatus collected from different localities(0%–3.7%).Conclusions: These findings demonstrated for the first time the establishment of COI gene for identifying I. granulatus nymphal tick which is of paramount importance to the control of potential tick-borne infections in Malaysia. Moreover, this study provides evidence that a combination of morphology and molecular data was corroborated as an accurate tool for tick identification.
