双重实时荧光PCR快速鉴别人参和西洋参＊

目的 建立一种能够快速鉴定人参和西洋参的双重实时荧光PCR方法。方法 通过对人参属及其近似种基因序列的分析比对，设计特异性的引物探针，建立双重实时荧光PCR检测方法。PCR反应体系为Premix Ex Taq （Probe qPCR） 10 μL，人参上下游引物各0.5 μL，西洋参上下游引物各0.3 μL，人参与西洋参特异性探针各0.4 μL，DNA模板2 μL，灭菌去离子水补至20 μL。反应条件为95℃预变性30 s；然后以95℃变性5 s，60℃退火延伸30 s进行40个循环。应用该方法测试了27份DNA样品，包括7份人参、6份西洋参、4份人参与西洋参混合样、10份其他人参属样品及其他常见中药材样品的DNA，以确定该方法的特异性。将人参与西洋参样品DNA混合后10倍稀释成不同浓度后进行检测，用以确定该方法的灵敏度。结果 人参及西洋参样品均在特定的荧光通道下出现典型的阳性扩增曲线，人参与西洋参混合样品均同时出现两条阳性扩增曲线，其它对照样品及空白对照均没有出现阳性扩增曲线。灵敏度检测结果显示该方法检测人参及西洋参的最低检测限均为2 pg DNA/反应。结论 本实验建立的双重实时荧光PCR方法能够同时快速、准确、灵敏地鉴别出人参和西洋参。     

基于线粒体COⅠ基因的广西不同地区福寿螺遗传多态性分析

目的 通过对广西不同地区福寿螺的COⅠ基因进行分析,以了解广西福寿螺种群的遗传多态性。方法 从南宁市横县、桂林市全州县和荔浦县、贺州市富川县、百色市田林县和崇左市凭祥县共计6个县区采集福寿螺样本,提取DNA并进行COⅠ基因的PCR扩增及测序。运用MAGE 7.0将本研究获得的单倍型与GenBank中的福寿螺单倍型使用邻接法构建系统进化树,并计算个体间的遗传距离,分析其遗传多样性。结果 COⅠ基因长度为493 bp,从11个样本鉴定出2种单倍型：单倍型1（Haplotype 1）和单倍型2（Haplotype 2）。其中9个样本为Haplotype 1,分别来源于南宁市横县（2个）、贺州市富川县（1个）、桂林市荔浦县（1个）、百色市田林县（2个）、崇左市凭祥县（3个）,该单倍型与小管福寿螺遗传距离最近,为0.047;2个样本为Haplotype 2,分别源自为南宁市横县和桂林市全州,与孤岛福寿螺遗传距离最近,为0.062。根据上述条件所构建的进化树形成了7个分支,分别为P. canaliculata、P. camena、P. insularum、P. paludosa、P. diffusa、P. haustrum、和外群Pilaconica。Haplotype 1与来自美国夏威夷 （GenBank登录号 EU 523129）的P. canaliculata组成一个分支,Haplotype 2与来自巴西（GenBank登录号EF514942）的P. insularum组成一个分支,且置信值均在98%以上。结论 初步判断广西地区存在小管福寿螺与孤岛福寿螺两种类型的福寿螺,其种群内没有发现遗传分化。     

Reopening schools in the context of increasing COVID-19 community transmission: The French experience

Background and objectivesThe role of schools in the spread of SARS-CoV-2 infections in the community is still controversial. The objective of our study was to describe the epidemiology of SARS-CoV-2 infections in different pediatric age groups during the first 2 months of the fall back-to-school period, in the context of increasing viral transmission in France.MethodsWeekly epidemiological data provided by Santé Publique France and the Ministry of National Education were analyzed according to the age groups defined by the different school levels. Weeks (W) 34–42 were considered for analysis.ResultsThe PCR positivity rate and incidence rate increased in all age groups during the study period, in an age-dependent manner. At W42, with adults being considered as reference, the risk ratio for a positive PCR test was 0.46 [95% CI: 0.44–0.49] and 0.69 [0.68–0.70] for children aged 0–5 years and 6–17 years, respectively. Similarly, the incidence rate ratio was 0.09 [0.08–0.09], 0.31 [0.30–0.32], 0.64 [0.63–0.66], and 1.07 [1.05–1.10] for children aged 0–5 years, 6–10 years, 11–14 years, and 15–17 years, respectively. Children and adolescents accounted for 1.9% of the newly hospitalized patients between W34 and W42, and for 1.3% of new intensive care admissions. No death was observed. Among infected children and adolescents, the percentage of asymptomatic individuals was 57% at W34 and 48% at W42. The number of schools closed remained low, less than 1% throughout the study period. The number of confirmed cases among school staff was consistent with the data measured in the general population.ConclusionIn the context of increasing viral transmission in the population, the spread among children and adolescents remained lower than that observed among adults, despite keeping schools open. However, the impact was age-dependent, with data in high schools close to those observed in adults.     

The Viral Load of Epstein-Barr Virus in Blood of Children after Hematopoietic Stem Cell Transplantation

ObjectiveTo detect the Epstein-Barr virus (EBV) viral load of children after hematopoietic stem cell transplantation (HSCT) using chip digital PCR (cdPCR).MethodsThe sensitivity of cdPCR was determined using EBV plasmids and the EBV B95-8 strain. The specificity of EBV cdPCR was evaluated using the EBV B95-8 strain and other herpesviruses (herpes simplex virus 1, herpes simplex virus 2, varicella zoster virus, human cytomegalovirus, human herpesvirus 6, and human herpesvirus 7). From May 2019 to September 2020, 64 serum samples of children following HSCT were collected. EBV infection and the viral load of serum samples were detected by cdPCR. The epidemiological characteristics of EBV infections were analyzed in HSCT patients.ResultsThe limit of detection of EBV cdPCR was 110 copies/mL, and the limit of detection of EBV quantitative PCR was 327 copies/mL for the pUC57-BALF5 plasmid. The result of EBV cdPCR was up to 121 copies/mL in the EBV B95-8 strain, and both were more sensitive than that of quantitative PCR. Using cdPCR, the incidence of EBV infection was 18.75% in 64 children after HSCT. The minimum EBV viral load was 140 copies/mL, and the maximum viral load was 3,209 copies/mL using cdPCR. The average hospital stay of children with EBV infection (184 ± 91 days) was longer than that of children without EBV infection (125 ± 79 days), P = 0.026.ConclusionEBV cdPCR had good sensitivity and specificity. The incidence of EBV infection was 18.75% in 64 children after HSCT from May 2019 to September 2020. EBV cdPCR could therefore be a novel method to detect EBV viral load in children after HSCT.     

Prospective case‐control study of enterovirus detection differences in children’s cerebrospinal fluid between multiplex PCR and real‐time RT‐PCR assay

BackgroundViral encephalitis is common in childhood. It is an acute brain parenchymal inflammation caused by a variety of viral infection, and enterovirus accounts for the majority. Due to atypical clinical manifestations, pathogenic testing is important for assisting clinical diagnosis. The purpose of this study was to evaluate the performance of the multiplex PCR assay compared with quantitative real‐time PCR for enterovirus detection.MethodsA prospective case‐control study was performed involving 103 pediatric patients suspected for viral encephalitis and cerebrospinal fluid (CSF) samples were collected and tested for 9 pathogens using multiplex PCR assay during April to November in 2018. In parallel, an aliquot of samples was tested for enterovirus infection by real‐time PCR assay.ResultsThere were 85.4% children were confirmed as viral encephalitis on discharge, the remaining ones were diagnosed as other CNS diseases, such as epilepsy. The specificity of the two methods was the same as that of the clinical diagnosis, but the sensitivity and consistency with clinical diagnosis of multiplex PCR were both higher than the real‐time PCR. Besides of enterovirus, multiplex PCR could also detect coinfection of enterovirus with Epstein‐Barr virus and mumps virus.ConclusionResults of multiplex PCR method are more consistent with the clinical diagnosis and are superior to real‐time PCR for detecting enterovirus in CSF.