常规RT-PCR与荧光定量RT-PCR检测GⅠ、GⅡ型诺如病毒方法的建立及其应用北大核心CSCD

目的建立GI、GII型诺如病毒的常规RT-PCR和荧光定量RT-PCR检测方法,并对两种方法进行应用。方法优化筛选出最佳PCR反应体系与反应条件,并从灵敏性、特异性、临床样品检测等方面对建立的方法进行比较与评价。结果该两种方法特异性强,与札幌病毒、轮状病毒、星状病毒、腺病毒同时检测无交叉反应,同一体系内GI、GII型诺如病毒相互之间没有干扰;常规RT-PCR最低检测限为103copies/μL,荧光定量RT-PCR最低检测限为102copies/μL;对180份临床粪便样品进行检测,常规RT-PCR则检测率为5.56%(10/180),符合率达97.22%,荧光定量RT-PCR检测率为8.33%(15/180),符合率达100%;对15份阳性样品测序分析,证实均为诺如病毒。结论建立的常规RT-PCR与荧光定量RT-PCR均可用于诺如病毒的快速检测,荧光定量RT-PCR更为灵敏。     

常规RT-PCR与荧光定量RT-PCR检测GⅠ、GⅡ型诺如病毒方法的建立及其应用

目的建立GI、GII型诺如病毒的常规RT-PCR和荧光定量RT-PCR检测方法,并对两种方法进行应用。方法优化筛选出最佳PCR反应体系与反应条件,并从灵敏性、特异性、临床样品检测等方面对建立的方法进行比较与评价。结果该两种方法特异性强,与札幌病毒、轮状病毒、星状病毒、腺病毒同时检测无交叉反应,同一体系内GI、GII型诺如病毒相互之间没有干扰;常规RT-PCR最低检测限为103copies/μL,荧光定量RT-PCR最低检测限为102copies/μL;对180份临床粪便样品进行检测,常规RT-PCR则检测率为5.56%(10/180),符合率达97.22%,荧光定量RT-PCR检测率为8.33%(15/180),符合率达100%;对15份阳性样品测序分析,证实均为诺如病毒。结论建立的常规RT-PCR与荧光定量RT-PCR均可用于诺如病毒的快速检测,荧光定量RT-PCR更为灵敏。     

贝类中GⅠ、GⅡ诺如病毒快速检测与分群方法的建立

目的建立贝类中GⅠ、GⅡ诺如病毒快速检测与分群方法。方法对比分析6株主要流行的GⅠ、GⅡ诺如病毒及参考诺如病毒的基因序列,筛选保守区域引物,优化建立SYBR Green Ⅰ荧光定量检测与熔解曲线快速分群方法,并对实际样品检测验证。结果引物P289/290可同时检测GⅠ、GⅡ诺如病毒,SYBR Green Ⅰ荧光定量检测方法在病毒浓度10~3～10~9 copies之间呈现良好的线性关系(R~2=0.993,P0.01),熔解曲线Tm值可区分GⅠ和GⅡ不同基因群的诺如病毒(F=7 507.60,P0.05)。120份贝类样品阳性检出率5.83%,其中阳性结果测序鉴定与快速分群方法结论一致。结论该方法成本低、检测快速、分群准确,具有良好重复性,适用于贝类中GⅠ、GⅡ诺如病毒的快速检测与分群。     

山东半岛地区贝类中诺如病毒污染状况与病毒鉴定初报

目的调查研究山东半岛沿海地区双壳贝类中诺如病毒污状况并对阳性株进行鉴定。方法应用常规RT-PCR与实时荧光定量RT-PCR,对2007年9月到2008年8月间在山东半岛沿海8个地区收集的186份双壳贝类样品进行了诺如病毒的定性和定量检测,并对阳性株核酸片段进行克隆、转化和序列分析,使用DNAStar软件进行核酸序列同源性比较,经MEGA4绘制系统进化树。结果 186份样品中检出5份诺如病毒阳性,检出率为2.67%,诺如病毒含量为大于102拷贝RNA,序列分析与遗传进化树显示5份诺如病毒阳性株均为GGII型NVs,与国内报道的GGII型毒株同源性达到98%以上。结论山东半岛地区贝类中含有的诺如病毒以GGII型为主,冬季检出率较高。     

贝类中GI、GII诺如病毒快速检测与分群方法的建立

目的 建立贝类中GI、GII诺如病毒快速检测与分群方法。 方法 对比分析6株主要流行的GI、GII诺如病毒及参考诺如病毒的基因序列,筛选保守区域引物,优化建立SYBR Green I荧光定量检测与熔解曲线快速分群方法,并对实际样品检测验证。结果 引物P289/290可同时检测GI、GII诺如病毒,SYBR Green I荧光定量检测方法在病毒浓度10³~10⁹ copies 之间呈现良好的线性关系(R²=0.993,P＜0.01),熔解曲线Tm值可区分GI和GII不同基因群的诺如病毒(F=7 507.60,P＜0.05 )。120份贝类样品阳性检出率5.83%,其中阳性结果测序鉴定与快速分群方法结论一致。 结论 该方法成本低、检测快速、分群准确,具有良好重复性,适用于贝类中GI、GII诺如病毒的快速检测与分群。     

张家界市某酒店因二次供水污染导致的诺如病毒感染性腹泻暴发疫情的调查

目的调查武陵源区某酒店一起游客诺如病毒感染性腹泻暴发疫情,查明原因,采取针对性措施,及时有效控制疫情。方法采用现场流行病学调查方法,描述病例分布和临床特征,分析危险因素,探讨其传播途径和方式,并对采集的标本进行实验室检测。结果本次疫情涉及到入住酒店的2个旅游团及部分酒店员工,共有病例33例,罹患率为29.73%,主要症状为腹泻、腹痛、恶心、呕吐;33份病例肛拭子标本中,12份检出诺如病毒GⅡ型阳性;自备水水池5份水标本中,全部检出诺如病毒GⅡ型阳性,菌落总数和大肠菌群超标;二次供水水塔5份水标本中,全部检出诺如病毒GⅡ型阳性,菌落总数和大肠菌群超标;早餐餐具涂抹10份标本中,2份检出诺如病毒GⅡ型阳性。结论本次疫情为一起由GⅡ型诺如病毒污染酒店二次供水引起的感染性腹泻暴发疫情。     

673例急性腹泻患儿诺如病毒GⅠ、GⅡ型感染快速筛查结果分析

目的?分析西安市儿童医院收治的急性腹泻患儿诺如病毒感染的流行病学特征，为西安市儿童诺如病毒防控提供科学依据。方法?采用胶体金法检测2021年1月—2022年12月西安市儿童医院收治的673例急性腹泻患儿的粪便样本诺如病毒GⅠ、GⅡ型。结果?673例样本中，诺如病毒检出率为20.51%（138/673），138例诺如病毒阳性样本中GⅠ型占34.06%（47/138）、GⅡ型占46.37%（64/138）、GⅠ型/GⅡ型占19.56%（27/138）；诺如病毒阳性样本集中在第1季度；男女急性腹泻患儿诺如病毒检出率分别为21.63%（85/393）、18.93%（53/280）；年龄＜3岁和≥3岁急性腹泻患儿的诺如病毒检出率之间差异有统计学意义（χ2＝3.852，P=0.001），其中0～6个月、7～12个月、1～岁、2～岁急性腹泻患儿诺如病毒检出率较高，依次为32.53%、30.48%、23.23%、30.43%；3～岁急性腹泻患儿主要发生诺如病毒GⅠ型/GⅡ型混合感染，占80%。结论?西安市急性腹泻患儿感染诺如病毒基因型主要是GⅡ型，春冬季节是高发期，年龄＜3岁婴幼儿是诺如病毒易感人群。     

厦门地区病毒性腹泻诺如病毒感染状况

目的了解本地区病毒性腹泻患者诺如病毒感染情况,为进一步探索诺如病毒流行规律和防控对策提供依据。方法收集2007年4月至2008年7月3个监测哨点医院病毒性腹泻患者粪便标本共323份,应用ELISA方法检测粪便标本中病毒抗原,实时荧光逆转录聚合酶链反应法(Real-Time RT-PCR)分组别检测病毒核酸,部分病毒核酸阳性的标本扩增RdRp基因片段后测序验证病毒组别。结果323例患者粪便标本中,ELISA检测抗原阳性68例,阳性率21.05%;Real-Time RT-PCR检测核酸阳性107例,阳性率33.13%;诺如病毒检出率为38.08%。诺如病毒GGⅡ组占74.77%,少数为GGⅠ组(1.87%),尚有25份(23.36%)未定型。结论厦门地区存在诺如病毒流行,以GGⅡ组毒株流行为主,而且是病毒性腹泻的主要病原。     

深圳市盐田区人群肠道病毒及常见感染性腹泻病毒调查分析

目的 了解深圳市盐田区人群肠道病毒及常见感染性腹泻病毒隐性感染状况,为制定防控措施提供科学依据。 方法 2020 年在盐田区海山、沙头角、盐田、梅沙四个街道,分别整群随机抽取 1 个社区为调查点,采集居民粪便样本用实时荧光定量 PCR 方法检测诺如病毒、札如病毒、轮状病毒、星状病毒、肠道腺病毒、肠道病毒通用型、肠道病毒 71 型(EV71)和柯萨奇病毒 A16 型(CV-A16)。 结果 共收集并检测粪便样品 813 份,未检出札如病毒、轮状病毒、星状病毒、肠道腺病毒、EV71 和 CA16;检出肠道病毒通用型 2 例,阳性率为 0. 25%,均为幼童口服脊髓灰质炎疫苗所致;检出诺如病毒 4 例,均为诺如 GⅡ型,阳性率为 0. 49%。 4 例诺如病毒阳性样本成功测序 2 例,分别为 GⅡ. 2-GⅡ. P16 型和 GⅡ. 17-GⅡ. P17 型,均与近年国内流行株有较高同源性。 结论 深圳市盐田区人群存在诺如病毒隐性感染,应加强对重点人群、重点场所的监测。     

5种尿液BK病毒DNA提取方法效果比较

目的探讨尿液BK病毒DNA的最佳提取方法。方法分别采用高速离心沉淀法、SDS煮沸裂解法、NaOH煮沸裂解法、PEG沉淀法和Tris饱和酚抽提法提取尿中BK病毒DNA进行PCR扩增,对不同方法BK病毒检出率结果进行比较。结果高速离心沉淀法、SDS煮沸裂解法、NaOH煮沸裂解法、PEG沉淀法和Tris饱和酚抽提法BK病毒检出率依次为78%（31/40）、100%（40/40）、83%（33/40）、95%（38/40）、100%（40/40）。结论在对BK病毒DNA的提取操作中,SDS煮沸裂解法灵敏度和安全性较高。     

Study on pollutant pathway of norovirus contamination in oysters

Noroviruses (NVs) cause human gastroenteritis through person-to-person transmission and via contaminated foods. In food poisoning, a major suspected cause is the consumption of raw oysters. We detected NVs from environmental water and oysters around a closed gulf where oysters are cultivated. We collected oyster and water samples once or twice a month for 30 months from October 2001 to March 2004. We then studied monthly changes in virus occurrence and in genetic relationships among 208 NVs isolated from water and oyster samples and from the feces of children suffering from acute gastroenteritis during the same period in the same region. In the analysis of untreated water flowing into farm sewage, NVs were detected year round. In other water samples -processed sewage, river water, and seawater-, oysters, and children's feces, NVs were detected mainly in winter. A comparison of NV nucleotide sequences showed genetic diversity, but some strains predominated in certain winter seasons. These predominant strains were detected across sample materials. In 2002/03, an identical strain was detected in sewage, river water, seawater, oysters, and feces. We also found that NV genetic types changed at the beginning of the season, in November or December, in both 2001/02 and 2002/03. This study showed a clear relationship between NVs detected in children's feces and those in environmental water and oysters. These results support the idea that NVs are transmitted from the feces of infected persons to oysters by the flow of water through farm sewage, rivers, and the sea, finally accumulating in the mid-gut gland of oysters.     

Occurrence of Giardia cysts and Cryptosporidium oocysts in activated sludge samples in Campinas, SP, Brazil

Giardia and Cryptosporidium have caused several outbreaks of gastroenteritis in humans associated with drinking water. Contaminated sewage effluents are recognized as a potential source of waterborne protozoa. Due to the lack of studies about the occurrence of these parasites in sewage samples in Brazil, we compared the efficiency of two procedures for concentrating cysts and oocysts in activated sludge samples of one sewage treatment plant. For this, the samples were submitted to i) concentration by the ether clarification procedure (ECP) and to ii) purification by sucrose flotation method (SFM) and aliquots of the pellets were examined by immunofluorescence. Giardia cysts were present in all samples (100.0%; n = 8) when using ECP and kit 1 reagents, while kit 2 resulted in six positive samples (85.7%; n = 7). As for SFM, cysts were detected in 75.0% and 100.0% of these samples (for kit 1 and 2, respectively). Regarding Cryptosporidium, two samples (25.0%; kit 1 and 28.5% for kit 2) were detected positive by using ECP, while for SFM, only one sample (examined by kit 1) was positive (12.5%). The results of the control trial revealed Giardia and Cryptosporidium recovery efficiency rates for ECP of 54.5% and 9.6%, while SFM was 10.5% and 3.2%, respectively. Considering the high concentration detected, a previous evaluation of the activated sludge before its application in agriculture is recommended and with some improvement, ECP would be an appropriate simple technique for protozoa detection in sewage samples.     

Multi-Walled Carbon Nanotubes for Magnetic Solid-Phase Extraction of Six Heterocyclic Pesticides in Environmental Water Samples Followed by HPLC-DAD Determination

Magnetic multi-walled carbon nanotubes were prepared as magnetic solid-phase extraction (MSPE) adsorbent for the enrichment of six heterocyclic pesticides in environmental water samples, including imidacloprid, triadimefon, fipronil, flusilazole, chlorfenapyr and fenpyroximate. Then six heterocyclic pesticides were separated and determined by high-performance liquid chromatography-diode-array detector (HPLC-DAD). Major factors influencing MSPE efficiency, including the dose of mag-multi-walled carbon nanotubes (mag-MWCNTs), extraction time, solution pH, salt concentration, type and volume of eluent and desorption time were investigated. Under the optimized conditions, the enrichment factor of the method reached to 250. The linearity was achieved within 0.05–10.0 μg/L for imidacloprid and chlorfenapyr, 0.10–10.0 μg/L for fipronil, flusilazole, triadimefon and fenpyroximate. Limits of detection were in the range of 0.01–0.03 μg/L. Good precision at three spiked levels were 1.1–11.2% (intra-day) and 1.7–11.0% (inter-day) with relative standard deviation of peak area, respectively. The developed method was utilized to analyze tap water, river water and reservoir water samples and recoveries at three spiked concentration levels ranged from 72.2% to 107.5%. The method was proved to be a convenient, rapid and practical method for sensitive determination of heterocyclic pesticides.     

2013 年安徽省农村环境卫生健康危害影响因素调查

目的 了解和掌握安徽农村改水改厕情况,垃圾、污水处理及媒介生物防治现状,为政府及相关行政部门制订农村环境卫生改善政策措施提供依据和支持。方法 按分层随机方式抽取24 个县,480个行政村和2 403 户,通过查阅资料、访谈、现场观察、实验室检测等方法获得监测数据。结果 2013 年安徽24 县农村集中式供水人口比例46.04%,卫生厕所普及率为35.9%。县级污水处理设施匮乏,且主要覆盖县城城区人口,农村地区大量生活、工业及养殖业污水随意排放,未能进行及时有效处理。大多数的调查村无专兼职保洁员,未开展“灭四害”工作。89.3%的调查户房屋周围有病媒生物孳生地。480 份土壤样品中有126 份检出蛔虫卵,检出率26.3%,其中有84 份检出活卵,活卵检出率17.5%;31.9%的镉含量超出国家二级土壤标准。结论 安徽农村环境卫生现状不容乐观,存在各种影响人群身体健康的危险因素,需进一步采取各种有效措施改善农村环境卫生现状。     

A waterborne outbreak of typhoid fever associated with a small drinking water supply system in Fuji city

Fifteen cases of typhoid fever occurred in "HARADA" district Fuji City in Shizuoka Prefecture during the period of 1983 to 1985. Epidemiological and bacteriological investigations were carried out on the samples from a small water supply system, and drains of apartment houses in 1985. Water from a small river in the neighboring water supply system, and faecal specimens from people living in close proximity were investigated. The results obtained were as follows: 1) Salmonella typhi were isolated from 3/3 patients (1 pupil and 2 employees) in 1983, 3/5 patients (3 employees) in 1984, and from 4/7 patients (1 kindergartner, 1 pupil and 2 junior high school boys) in 1985. Phage type of these isolates were the same type "D1". 2) In the bacteriological survey on environment, S. typhi (phage type D1) were isolated from water of the water source of "HARADA" water supply in Fuji City with membrane filter methods, and from the sand source "HARADA" water supply with enrichment culture methods. Also, with an examination that tested 3,670 inhabitants, it was found that a carrier (phage type D1) was living near the "HARADA" water supply. 3) In the three selective media for the isolation of S. typhi, Bismuth sulfite agar (Difco) was the most sensitive, and detected 13 samples (61.9%) out of 21, from water source and sewage.     

Pathogenic Acanthamoeba strains from water sources in Jamaica, West Indies

In 2004, samples of tap water and of river and sea water associated with human activities were collected in Jamaica, West Indies, and checked for free-living Acanthamoeba. The morphologies of the cysts and trophozoites observed and the results of PCR-based amplifications with a genus-specific primer pair were used to identify the Acanthamoeba isolates. The potential of each isolate as a human pathogen was then evaluated using thermotolerance and osmotolerance assays and two PCR-based assays for Acanthamoeba pathogenesis. Acanthamoeba were identified in 36.1%, 26.4% and 49.6% of the tap-, river- and sea-water samples collected, respectively. Pathogenic potential was shown by 60.0% of the Acanthamoeba strains isolated from tap water, 68.4% of the strains from river water, and 40.4% of the seawater strains. Sequencing of ribosomal DNA revealed the T1, T2, T4, T5, T7, T9 and T11 genotypes. Isolates of the T4 genotype were collected from tap, rain and sea water and, as expected, exhibited the most pathogenic traits; most were osmotolerant, thermotolerant and expressing extracellular serine protease.This is the first study of the occurrence and distribution of Acanthamoeba in water in the West Indies, and the results confirm the presence of potentially pathogenic strains in Jamaica.     

Isolation and incidence of Cryptosporidium and Giardia from river water

A study was conducted during the period from July to October in 1999, surface water samples were collected from 13 rivers in Hyogo Prefecture serving as tap water sources and examined for Cryptosporidium oocysts and Giardia cysts using the immunomagnetic separation method. In 9 (69%) of the 13 rivers, Cryptosporidium oocyst was detected. Giardia cyst was detected in 5 (38%) of the 13 rivers. The Cryptosporidium oocyst positive rate was comparable to that of fecal bacteriological indicators, which was also examined and detected in 10 (77%) of 13 rivers. Water samples were collected at 69 points in the 13 rivers. Cryptosporidium parvum oocyst was detected at 38 (55%) of the 69 points, demonstrating widely spread contamination. Comparing 3 areas which were divided on the basis of agricultural circumstance as well as geographical locating, the Cryptosporidium positive rate varied widely according to the area, ranging from 37% to 100%. To elucidate the reason for these differences, the relationship between positive rate and the species and number of livestock animals raised in each respective region was investigated. The results showed a strong correlation (r = 0.91) between the number of raised cattle and contamination with the rate of Cryptosporidium in each area. Furthermore, genetic analysis by PCR-restriction fragment length polymophism method revealed that C. parvum oocyst detected in the rivers studied was the bovine type, probably indicating that the oocyst was excreted from the cattle in the river basins. The degree of contamination with Cryptosporidium in river water was comparable to that of fecal bacteriological indicators, suggesting the possibility that fecal bacteriological indicators examination might be used as a criterion of Cryptosporidium contamination.     

Outbreak of cryptosporidiosis after contamination of the public water supply in Saitama Prefecture, Japan, in 1996

An outbreak of cryptosporidiosis occurred in Ogose Town, Saitama Prefecture. Japan, in June 1996. Of 12,345 respondents to a questionnaire sent to households in the town (population; 13,809), 8,812 (71.4%) reported an acute gastrointestinal illness some time between May and July. In addition, 274 traceable visitors at local inns, golf courses, and the like during this period and 54 employees from out of town were infected. Cases of cryptosporidiosis were estimated to 9,140. Of these, 2,856 subjects were treated at outpatient clinics and 24 subjects were hospitalized (some subjects counted twice). No deaths were attributed to the outbreak. Among the visitors to Ogose who were traced, 7 persons who stayed only one day during the outbreak and drank half a glass to 2 glasses (100 to 360 ml) of tap water had cryptosporidiosis confirmed by laboratory tests. The median incubation period for the 14 persons for whom this calculation was possible was 6.4 days (range, 5 to 8 days). Of 469 pupils reporting details of their fever and diarrhea, abdominal cramps, or these combined signs and symptoms, the median maximum body temperature was 37.8 degrees C (range, 36.7 to 40.3 degrees C). The duration of illness, reported by 608 of the pupils, was 5.2 days (range, 1 to 15 days), and that reported by 187 employees was 4.8 days (range, 1 to 18 days). The longest known time for discharge of oocysts after onset was 44 days. Blood was not found in the 609 stool specimens examined. The outbreak was caused by contamination of the town's potable water by Cryptosporidium parvum oocysts. The town's water treatment plant treated river water by coagulation, sedimentation, sand filtration, and chlorination. Contamination arose because of various natural and artificial factors: one was that the monthly precipitation in May was much lower than average, causing the river water level to drop. Another factor was heavy rainfall one night in May that increased water turbidity. The amounts of the coagulant added seemed to be insufficient. There are two inns, three public lavatories, and two small-scale wastewater treatment plants upstream 400 m and 1,200 m of the intake point of the town's water treatment plant. However, there are no farms with livestock in the area. We suggest that the location of the water treatment facilities was inappropriate, and that oocysts had circulated from the potable water to humans to sewage to the river and back to the potable water.     

Identification of HEV in symptom‐free migrants and environmental samples in Italy

Hepatitis E virus (HEV) is considered an emerging pathogen in industrialized countries. The occurrence of HEV genotypes in samples of faeces from asymptomatic migrants arriving on the coasts of South Italy and environmental samples was investigated. Analyses of sequences were used to compare human and environmental genotypes. A total of 40 stool specimens, 12 samples of untreated urban sewage, 12 samples of treated urban sewage and 12 samples of surface water were analysed. Viruses were concentrated from water samples by the tangential flow ultrafiltration technique. The presence of HEV RNA was detected by nested RT‐PCR. Viral isolates were sequenced and phylogenetically characterized. Two (5%) of the 40 faecal samples tested were found to be positive for HEV RNA (G1 and G3 genotypes). The virus was detected in 25% (3/12) of the untreated sewage samples and 25% (3/12) of the surface water samples: all isolates belonged to G3 genotype. None of the treated sewage samples were found to be HEV RNA positive. The virus was detected in the faeces of two asymptomatic subjects, suggesting a potential role for symptom‐free HEV carriers as a human reservoir. G3 HEV strains were detected in the untreated sewage, as observed in similar studies conducted in other European countries but differing from another study conducted in Italy recently. Moreover, our results show the first case of HEV isolated from fresh surface waters.