Improvement in HLA-DQB typing by PCR-RFLP: Introduction of a constant restriction site in one of the primers for digestion control

The PCR-RFLP method previously reported by Inoko is a powerful technique for HLA class II typing. The reliability of RFLP interpretation depends on complete digestion by restriction endonucleases using a modified primer with restriction sites as an internal digestion control. The use of restriction enzymes which recognize specific HLA DQB allelic variations makes HLA DQB genotyping possible.     

Polymorphism of the human retinoid X receptor beta and linkage disequilibrium with HLA-DPB1

The human retinoid X receptor beta (RXRB) gene is localized in the major histocompatibility complex (MHC) region between DPB1 and RING2. The RXRB gene sequence reported by different investigators suggests that the gene may be polymorphic. In this study, we confirmed one polymorphism by sequencing genomic DNA from four Caucasian individuals. We also developed a restriction fragment length polymorphism (RFLP) analysis to detect this specific polymorphism. Linkage analysis studies between RXRB alleles and a number of HLA markers showed significant linkage disequilibrium between RXRB*T and HLA-DPB1*0401.     

Genotyping of Hepatitis C virus (HCV) in infected patients from South India

Hepatitis C virus shows substantial nucleotide sequence diversity distributed throughout the viral genome. In the present study genotyping for Hepatitis C virus (HCV) infected patients was based on RFLP analysis of 5' UTR and using type specific primers of NS5B regions. It was observed that 60% of the patients (30 patients with chronic hepatitis) were infected with variants of genotype 1 and 40% of the patients (4 chronic hepatitis patients, 12 patients with chronic renal failure and 4 cirrhosis) were infected with variants of type 3 of HCV. None of the cirrhotic patients and patients with chronic renal failure, in the present study, were infected with type 1 of HCV. While PCR-RFLP, typing was rapid in conjunction with the primers used for RT-PCR, NS5 typing was helpful in determining the subtype. There was good correlation between the two typing methods and this method can be used as a cost-effective method for studying large number of samples. The study shows that predominant genotypes of HCV in South India include type 1 and 3. Type 3 seems to be transmitted nosocomially as suggested by the results in patients with chronic renal failure, as these patients are exposed to multiple medical interventions.     

HSP70-hom基因多态性与高原反应易感性的关系

目的 探讨热应激或热休克蛋白70-hom(HSP70-hom)基因型与高原反应易感性间的关系,为发现和保护易感人群提供依据。方法随机选取武警战士229人,其中56人有高原反应者为病例组,173人无高原反应者为对照组。应用聚合酶链反应～限制性片段长度多态性(PCR-RFLP)分析方法,研究两组观察对象中HSP70-hom的基因型分布情况。结果病例组HSP70-hom A／A基因型的频率明显高于对照组,差异有显著性(P＜0．05)。结论 HSP70-hom A／A基因型的机体可能存在应激能力较弱的问题,这有助于在特定职业人群中发现和保护这些个体,为保障健康,提高工作效率提供科学依据。     

贵州省2004年分离到的脊髓灰质炎病毒分子生物学特征

目的研究贵州省2004年分离到的脊髓灰质炎(脊灰)病毒分子生物学特征。方法对贵州省2004年分离到的所有脊灰病毒,用聚合酶链反应-限制性酶切片段长度多态性分析(PCR-RFLP)和酶联免疫吸附试验(ELISA)法进行了型内鉴定,并对型内鉴定异常株进行了VP1区的序列测定。结果贵州省在2004年急性弛缓性麻痹(AFP)病例及接触者、流动人口和健康儿童的粪便标本中,共有95例分离到脊灰病毒。其中Ⅰ型22例,Ⅱ型26例,Ⅲ型21例,混合型19例,脊灰病毒混合非脊灰肠道病毒7例。经用PCR-RFLP和ELISA方法进行型内鉴定,共有16株病毒与疫苗株病毒存在差异,其中3株脊灰病毒与疫苗株病毒在PCR-RFLP图谱上有差异[其中1株同时为双反应(DRV)],3株ELISA结果为DRV,11株ELISA结果为非疫苗类似株(NSL)。在这些型内鉴定异常株病毒中,Ⅰ型13株,Ⅱ型3株。对这16株脊灰病毒进行VP1区序列测定,发现9株Ⅰ型疫苗衍生脊灰病毒(VDPV)和1株Ⅱ型VDPV。结论根据对贵州省2004年从95例AFP病例及接触者、流动人口、健康儿童分离的脊灰病毒的血清定型结果和型内鉴定结果及对13株Ⅰ型和8株Ⅱ型脊灰病毒VP1区核苷酸序列测定证实,脊灰减毒活疫苗病毒在人群的循环导致疫苗病毒神经毒力恢复突变。通过AFP病例监测系统及时发现了Ⅰ型VDPV的循环和Ⅱ型VDPV。对2004年下半年脊灰病毒基因特点的分析,提示贵州省已经阻断了Ⅰ型VDPV的循环。     

Molecular Detection and Identification of Theileria Species by PCR-RFLP Method in Sheep from Ahvaz,Southern Iran

Background

The present study was carried out to investigate the accurate status of ovine Theileria infection in sheep from Ahvaz and surrounding region, a tropical area southwest Iran.

Methods

A PCR-RFLP method based on 18S ribosomal RNA gene was designed which could detect and differentiate Theileria and Babesia spp. and also differentiate main Theileria species in sheep at the same time. 119 sheep blood samples were collected from Ahvaz and surroundings.

Results

Microscopic examination of blood smears revealed 69.7% (83/119) infection with Theileria spp. Of the total samples subjected to PCR, 89% (106/119) were found to be positive, all of which were identified as Theileria by RFLP analysis using enzyme Hind II. In enzymatic digestion of PCR products by Vsp I, 91.5% (97/106) of Theileria positive samples were identified as T. ovis while mixed Theileria infections were found in 9 samples. The samples with mixed infections were analyzed with an additional nested PCR-RFLP method, by HpaII enzyme digestion. 3 samples with T. lestoquardi infection, 1 sample with T. ovis and T. annulata, 1 sample with T. lestoquardi and T. annulata, and 4 samples with T. ovis, T. lestoquardi and T. annulata mixed infections were detected.

Conclusion

Ovine theileriosis caused by T. ovis is highly prevalent in southwest Iran while T. lestoquardi and T. annulata infection can be detected in a lesser propor-tion of sheep in this region. The new PCR-RFLP method that was designed in this study, can serve as a beneficial diagnostic tool, especially in T. ovis prevalent re-gions.     

Complete HLA-A DNA typing using the PCR-RFLP method combined with allele group- and sequence-specific amplification

We have established a practical method of complete high-resolution typing for all HLA-A alleles using the polymerase chain reaction (PCR)-restriction fragment-length polymorphism (RFLP) technique combined with allele group- and sequence-specific amplification. The second and third exons of the HLA-A gene, in which most allelic variations are observed, were separately amplified by PCRs with 3 and 4 group-specific primer pairs, respectively. Each PCR-amplified product was digested by allele-specific restriction endonucleases and then subjected to electrophoresis on a 10% polyacrylamide gel. In this way, 62 out of 79 HLA-A alleles could be discriminated by the RFLP patterns derived from the genetic polymorphism in the exon 2 and 3 domains. The remaining 17 alleles could be defined unequivocally by either PCR-RFLP analysis after exon 4 amplification or PCR analysis with sequence-specific primers (SSP). By this method, complete HLA-A genotyping for all homozygous and heterozygous combinations can be accomplished, establishing technically simple, economical and practical routine typing of the HLA-A gene, especially for small samples.     

新疆水源性隐孢子虫的检测

目的检测新疆15个城市自来水和河水隐孢子虫污染情况。方法采集水样,分别应用改良美国环保局(EPA)1622法及巢式PCR(nested-PCR)方法进行检测。1)改良EPA1622法:水样经微孔滤膜抽滤、淘洗,磁抗体分离法分离纯化后免疫荧光染色鉴定。2)Nested-PCR法:用试剂盒提取纯化的隐孢子虫卵囊基因组DNA,针对隐孢子虫小亚单位核糖体RNA(18SrRNA)部分基因,依据文献设计并合成引物,用巢式PCR扩增,产物纯化后经SspⅠ及VspⅠ单酶切,并进行RFLP分析。结果2种方法检测新疆15个地区的自来水隐孢子虫卵囊均为阴性,改良EPA1622法检测乌鲁木齐市、昌吉市、伊宁市和吐鲁番市的河水隐孢子虫卵囊阳性。巢式PCR检测乌鲁木齐市和伊宁市水样,均扩增出约830 bp的特异片段,RFLP初步鉴定为小鼠隐孢子虫基因;昌吉市和吐鲁番市河水水样PCR检测阴性。结论新疆乌鲁木齐市、伊宁市河水检出隐孢子虫,初步鉴定为小鼠隐孢子虫。而当地的饮用水未受污染。     

Identification and phylogenetic relationship of Iranian strains of various Leishmania species isolated from cutaneous and visceral cases of leishmaniasis based on N-acetylglucosamine-1-phosphate transferase gene

The identity of Iranian Leishmania species has been resolved to some extent by some genetic markers. In this study, based on N-acetylglucosamine-1-phosphate transferase (nagt) gene, we further elucidated the identity and phylogeny of the prevalent species in this country. DNAs of 121 isolates belonging to cutaneous leishmaniasis (CL) patients, canine visceral leishmaniasis (CVL) cases, and Rhombomys opimus rodents were amplified by targeting a partial sequence of nagt gene. All the amplicons were analyzed with restriction fragment length polymorphism (RFLP) using Acc1 enzyme, and 49 amplicons representing different reservoir hosts were sequenced and aligned with similar sequences from GenBank database. The RFLP analysis revealed that 41 CL patients were infected Leishmania tropica and 36 with Leishmania major. Among 10 CVL isolates, 6 were identified as Leishmania infantum and 4 as L. tropica. Amongst 34 rodents’ isolates, 11 and 23 isolates exhibited patterns similar to those of L. major, and L. tropica/Leishmania turanica, respectively. The sequencing results from all CL patients, CVL cases, and 4 reservoir rodents were in agreement with RFLP analysis and showed 99–100% homologies with the registered species of L. major, L. tropica, and L. infantum from Turkey, Tunisia, Iraq and Israel. Of the 7 rodent isolates exhibiting RFLP patterns similar to L. tropica/L. turanica, 3 exhibited the highest homologies (99–100%) with L. turanica and 4 with Leishmania gerbilli. The 49 nagt DNA sequences were grouped into five clusters representing L. major, L. tropica, L. infantum, L. turanica and L. gerbilli species, encompassing 19 haplotypes. No correlation was observed between intraspecies divergence and geographic distribution of haplotypes. The L. tropica haplotypes exhibited more homologies with those of L. infantum than L. major (97.2% vs. 96.9%), a probable indication to the potential ability of L. tropica to visceralize. Characterization of Iranian Leishmania isolates using nagt gene allowed unambiguous identification of five prevalent species with a high-resolution phylogeny.