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。     

Study of hepatitis B virus gene mutations with enzymatic colorimetry-based DNA microarray

OBJECTIVES: To establish a modified microarray method for detecting HBV gene mutations in the clinic. DESIGN AND METHODS: Site-specific oligonucleotide probes were immobilized to microarray slides and hybridized to biotin-labeled HBV gene fragments amplified from two-step PCR. Hybridized targets were transferred to nitrocellulose membranes, followed by intensity measurement using BCIP/NBT colorimetry. RESULTS: HBV genes from 99 Hepatitis B patients and 40 healthy blood donors were analyzed. Mutation frequencies of HBV pre-core/core and basic core promoter (BCP) regions were found to be significantly higher in the patient group (42%, 40% versus 2.5%, 5%, P < 0.01). Compared with a traditional fluorescence method, the colorimetry method exhibited the same level of sensitivity and reproducibility. CONCLUSIONS: An enzymatic colorimetry-based DNA microarray assay was successfully established to monitor HBV mutations. Pre-core/core and BCP mutations of HBV genes could be major causes of HBV infection in HBeAg-negative patients and could also be relevant to chronicity and aggravation of hepatitis B.     

Novel mutations in the cytochrome P450 2C19 gene: a pitfall of the PCR-RFLP method for identifying a common mutation

CYP2C19 is a clinically important enzyme involved in the metabolism of therapeutic drugs such as (S)-mephenytoin, omeprazole, proguanil, and diazepam. Individuals can be characterized as either extensive metabolizers (EM) or poor metabolizers (PM) on the basis of CYP2C19 enzyme activity. The PM phenotype occurs in 2–5% of Caucasian populations, but at higher frequencies (18–23%) in Asians. CYP2C19*2 and CYP2C19*3, which are single-nucleotide polymorphisms of CYP2C19, are the main cause of PM phenotyping in homozygotes or compound heterozygotes. We report two novel mutations in the CYP2C19 gene identified by direct sequencing and subcloning procedures. One of these mutations was considered to be CYP2C19*3 by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). This result suggests that mutations classed as CYP2C19*3 might include other mutations. Further studies are needed to clarify the relationship between these novel mutations and enzyme activity.The DDBJ accession number of the novel mutation is AB113829     

Epidemiologic Study of Malassezia Yeasts in Seborrheic Dermatitis Patients by the Analysis of 26S rDNA PCR-RFLP

Background

This case-control study concerns a molecular biological method based on the data gathered from a group of Korean subjects to examine the distribution of Malassezia yeasts in seborrheic dermatitis (SD) patients. Cultures for Malassezia yeasts were taken from the foreheads, cheeks and chests of 60 patients with SD and in 60 healthy controls of equivalent age.

Objective

The purpose of this study is to identify the relationship between certain species of Malassezia and SD. This was done by analyzing the differences in the distribution of Malassezia species in terms of age and body parts of the host with healthy controls.

Methods

26S rDNA PCR-RFLP, a fast and accurate molecular biological method, was used to overcome the limits of morphological and biochemical methods.

Results

The positive Malassezia culture rate was 51.7% in patients with SD, which was lower than that of healthy adults (63.9%). M. restricta was dominant in patients with SD (19.5%). Likewise, M. restricta was identified as a common species (20.5%) in healthy controls. In the ages 31~40, M. restricta was found to be the most common species (31.6%) among SD patients.

Conclusion

According to the results of the study, the most frequently isolated species was M. restricta (19.5%) in patients with SD. There was no statistically significant difference in the distribution of Malassezia species between the SD patients and healthy control groups.     

雌激素受体α基因多态性与子宫内膜异位症的相关性

目的探讨雌激素受体α（Estrogen receptorα ERα）基因多态性与子宫内膜异位症（Endometriosis EM）发病的相关性。方法选取107例经开腹或腹腔镜手术治疗后,病理确认为子宫内膜异位症的患者作为实验组,选取健康体检者80例作为正常对照组。提取DNA,采用聚合酶链式反应-限制性片段长度多态性（Polymerase chain reaction-restriction fragment length polymor-phism PCR-RFLP）的方法,检测ERα基因XbaⅠ和PvuⅡ位点多态性。结果 ERα基因多态性显示ERα-XbaⅠ基因多态性在子宫内膜异位症组与对照组间的分布则有显著性差异（P=0.039）,XX/Xx样本组51（47.66%）,对照组25（31.25%）,两者有显著性差异（P=0.024）。X等位基因患EM的风险是x等位基因的2.00倍（OR=2.00,95%CI：1.09-3.67）。而ERα-PvuⅡ基因多态性在EM与对照组间的分布差异无显著性,（P=0.285）。结论 ERα-XbaⅠ基因多态性在子宫内膜异位症组与对照组间的分布则有显著性差异（P=0.039）。提示ERα-XbaⅠ基因多态性与EM发病相关。     

白细胞介素-10基因多态性与子宫内膜异位症的相关性

目的探讨白细胞介素-10（Interleukin-10,IL-10）基因多态性与子宫内膜异位症（Endometriosis EMs）发病的相关性。方法107例经手术治疗后,病理确认为EMs的患者,术中留取新鲜组织,对照组80例,取全血,提取DNA,采用聚合酶链式反应-限制性片段长度多态性（Polymerase chain reaction-restriction fragment length polymorphism PCR-RFLP）的方法,检测IL-10启动子区域-1082、-819、-592位点多态性。结果EMs组与对照组的IL-10启动子区域-1082、-819、-592基因型分布差异均无统计学意义（P〉0.05）。结论IL-10基因多态性与EMs的发病无明显相关性,未能够证实IL-10等位基因与EMs的遗传易感性有关。     

Distribution of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutant alleles in Plasmodium vivax isolates from Thailand

The analysis of prevalence and distribution of pvdhfr and pvdhps mutations were performed in 169 samples collected from patients with Plasmodium vivax infection who attended the malaria clinics in the provinces along the three international borders of Thailand (Thai-Myanmar, Thai-Cambodian, and Thai-Malaysian borders). SNP-haplotypes of the pvdhfr at amino acid positions 13, 33, 57, 58, 61, 117, and 173 and of the pvdhps at positions 383 and 553 were examined by nested PCR-RFLP. Significant differences in the prevalence and distribution of pvdhfr and pvdhps combination alleles were observed in P. vivax isolates collected from all the three border areas. The most prevalent combination alleles were triple mutant pvdhfr 57L/58R/117T alleles/double wild-type pvdhps alleles (n = 18), double mutant pvdhfr 58R/117N alleles/double wild-type pvdhps alleles (n = 10), and triple mutant pvdhfr 58R/61M/117N alleles/double wild-type pvdhps alleles (n = 52) or with single mutant pvdhps 383G allele (n = 28), respectively. These information on prevalence and patterns of pvdhfr and pvdhps polymorphisms obtained from the present study suggest the presence of SP pressure on P. vivax isolates in Thailand which could be linked to the introduction of malaria from neighboring countries. Results did not support the application of SP for P. vivax control program in Thailand as well as the neighboring countries.     

HindII and SduI digests of heat-shock protein 70 PCR for Leishmania typing

Restriction fragment length polymorphisms of the heat-shock protein 70 gene have been used for discriminating Leishmania species. Here, we validated HindII as a much cheaper alternative to EcoRII and SduI for discriminating Leishmania (Viannia) braziliensis from Leishmania (Viannia) naiffi and an atypical Leishmania (V.) braziliensis group, which was previously not possible.     

脱落细胞线粒体基因A1555G突变在诊断线粒体耳聋中的应用

目的用PCR-RFLP技术检测脱落细胞(尿液沉渣细胞和颊黏膜细胞)线粒体基因A1555G突变,探讨脱落细胞用于诊断线粒体基因突变相关耳聋的可行性。方法收集福建省某特殊教育学校126名聋哑学生和1个线粒体基因A1555G突变的遗传性耳聋家系6例患者外周血及尿液沉渣细胞和颊黏膜细胞,用PCR-RFLP技术筛查患者是否携带线粒体DNA A1555G突变,并与测序法进行比较。结果尿液沉渣细胞检测线粒体DNA A1555G突变的检出率最高(9/132),颊黏膜细胞和外周血细胞均为7/132。PCR-RFLP筛查结果与直接测序法基本相符。结论脱落细胞适用于耳聋相关线粒体DNA A1555G突变检测。     

Identification and characterization of 31 isolates of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> (Spirochaetales,Spirochaetaceae) obtained from various hosts and vectors using PCR-RFLP and SDS-PAGE analysis

Borrelia burgdorferi sensu lato, the etiologic agent of Lyme borreliosis, circulates between ticks and vertebrate hosts. Two main genospecies typically occur in the Czech Republic Borrelia garinii and Borrelia afzelii, transmitted generally by Ixodes ricinus (L., 1758) ticks. The aim of our study was to identify spirochaete isolates focusing on Borrelia burgdorferi acquired from different sources: vectors (ticks), potential vectors (mosquitoes, small mites) and hosts (wild rodents). In the years 1996–2001 a total of 2398 ticks, 72 mites (from wild rodents), 2700 mosquito adults, 1798 mosquito larvae and organ parts (kidney and spleen) of 216 wild rodents were collected from seven localities in the Czech Republic. A total of 31 spirochaete strains were isolated: 13 strains from ticks, 1 strain from mite (Haemogamasus sp.), 15 strains from rodents, 1 strain from mosquito adults and 1 strain from mosquito larva. For the genospecies identification of these isolates PCR, PCR-RFLP was used and their characterization was also performed by SDS-PAGE. By nested PCR method all except one isolated strains were detected as Borrelia burgdorferi s.l. Following PCR-RFLP molecular analysis results, tick isolates were identified as B. garinii and B. afzelii, the strain isolated from the mite was identified as B. afzelii. This is the first isolated strain of B.b.s.l. from a different mite of infraorder Parasitiformes than tick. All of rodent isolates were identified as B. afzelii; mosquito adult isolate was identified as B. afzelii. Larval isolate from mosquito is spirochaete, but does not belong to Borrelia burgdorferi sensu lato group.