深圳地区柯萨奇病毒A组16型的分子流行病学研究及RT-LAMP检测方法的建立
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摘要
研究背景和目的
儿童手足口病(hand-foot-mouth disease, HFMD)是一种常见的传染性强、传播途径复杂、在短时间内可造成较大规模流行病毒感染性疾病,其主要病原体是柯萨奇病毒A组16型(Coxsackievirus A16, CA16)和肠道病毒71型(Enterovirus71,EV71)。大多数HFMD患者症状轻微,主要以手,足、口等部位皮肤粘膜的疱疹、溃疡为主要特征,伴有全身症状如发热、厌食、乏力、精神萎靡等。少数患儿可出现中枢神经、呼吸系统损害,引发脑炎、急性弛缓性麻痹、脑水肿、肺水肿和心肌炎等,个别重症患儿病情进展快,易发生死亡。EV71由于能引起严重的神经系统相关症状而得到高度重视,对其研究也较为深入。相反,CA16却由于引起较轻的临床症状不受关注,一直被作为需要与EV71鉴别诊断的病毒附带进行研究,流行病学资料很少,也没有统一的基因分型标准。然而,近年已有报道指出CA16感染可能与心肌炎、心包炎及其它严重疾病有关,甚至还可引起成人致死性肺炎。因此,开展CA16感染的流行病学研究,可为防控CA16病毒引起的相关疾病提供基础信息。基于此,本研究拟对2005-2009年期间深圳市的HFMD患者进行CA16病原学检测,对CA16分离株VPl区进行核苷酸序列测定,并与国内外CA16毒株进行同源性分析,旨在了解深圳地区CA16病毒的流行情况及基因型特征,以期为HFMD的综合防治和CA16病毒的动态监测提供依据,并丰富CA16毒株基因库及分子流行病学资料。
CA16常规检测方法主要有病毒分离、免疫组化、中和试验及逆转录聚合酶链反应(RT-PCR).经典的细胞分离培养、特异性抗体检测方法步骤烦琐,检测周期长,而且一些肠道病毒难以在细胞中进行增殖,容易出现漏检；基于CA16核酸扩增的分子生物学诊断技术如PCR、RT-PCR、Real-Time RT-PCR (real-time fluorescent RT-PCR)等在病原微生物的检测以及疫病诊断方面发挥着重大作用,克服了以上缺点,敏感度、特异性较高,已成为目前CA16快速诊断的重要手段,但这些方法或者需要精密控温设备和高级复杂的分析仪器,或者对操作人员的熟练度和专业水平要求比较高,且反应时间长,不利于现场样品的检测,不利于在基层推广,无法满足简单、快速、准确诊断的要求,而疫病的防控和治疗的关键在于对病原微生物的快速、准确、及早的检测并确诊。因此,建立一种快速、准确的新型诊断技术对于CA16的诊断就显得尤为重要。
环介导等温扩增(Loop-mediated isothermal amplification, LAMP)技术是Notomi等2000年建立的一种新的核酸扩增方法。其原理是利用一种链置换DNA聚合酶(Bst DNA polymerase)和两对特殊的引物,特异地识别靶序列上的6个独立区域,在等温条件下(65℃左右)保温几十分钟,即可完成核酸扩增反应。反应结果可直接靠扩增副产物焦磷酸镁的沉淀浊度进行判断,也可以由添加SYBR Green I、溴化乙啶(EB)或其它核酸染剂进行呈色后观察。其优点是特异性强,扩增效率高,反应灵敏,操作简单。LAMP技术目前已被国内外学者应用到多种细菌及病毒的基因检测中,但至今尚未检索到使用该技术检测CA16的报道。本研究拟建立CA16病毒逆转录环介导等温扩增(RT-LAMP)技术,并比较该技术与RT-PCR及Real-Time RT-PCR对深圳市临床诊断HFMD患者标本的CA16基因检测效果,旨在为临床CA16感染诊断寻求快速、简便、可行的新方法。
方法
1．CA16病毒分子流行病学特征
首先用荧光RT-PCR方法对深圳地区2005-2009年5年间所有HFMD病例进行CA16病毒快速检测,再选取每年具有代表性的阳性株用CA16特异性引物对VPl基因进行RT-PCR扩增,阳性扩增产物用于核苷酸序列测定,所得的序列与CA16各基因型代表株进行比较,用DNASTAR和Mega 4.1软件进行种系进化分析。
2.CA16病毒RT-LAMP检测方法的建立
首先针对CA16VP1区保守序列设计RT-LAMP引物,对引物进行反复筛选,选择最优引物组合,其次对反应体系进行优化,再对优化的RT-LAMP体系进行灵敏性、特异性、重复性实验验证,并运用于临床样本检测,初步评价CA16 RT-LAMP检测技术的可操作性。
结果：
1．CA16病毒分子流行病学特征分析
2005年～2009年共收集HFMD患者标本1161份,CA16检出占HFMD患者的21.4%,每年的检出率分别为：2005年27.0%,2006年51.6%,2007年27.6%；2008年17.8%；2009年17.0%。主要引起5岁以下儿童(92.7%)的感染,男女性别之间发病差异无统计学意义(P=0.469),发病高峰为每年的夏秋季节,病例大多集中在3-6月,9-11月还有一次小流行,龙岗区、宝安区(郊区)的发病显著高于福田区、南山区、罗湖区、盐田区(城区)。
本研究选取的69株CA16深圳分离株VP1区核苷酸和氨基酸同源性分别为96.8%和99.9%。基于25株深圳代表株与38株国内国际参考株的比较分析,参考EV71分型标准及Perera等的CA16分型标准,CA16可分为A、B两种基因型,B型可分为B1、B2亚型,B2亚型又可分为B2a、B2b、B2c基因亚群,之前报道的B和C基因型应归于同一B基因型下二个分支,B基因型应该归于B1基因亚型,C基因型应该归于B2基因亚型。各基因型间氨基酸变异位点分析未发现特异性突变。2005-2009年深圳分离株在CA16进化树上分别位于B2a(66.7%)和B2b(33.3%)分支上,且与同一时期同一基因型别国内国际CA16分离株的同源性较高。
2.CA16病毒RT-LAMP检测方法
建立的RT-LAMP检测方法快速、简便、成本低,反应在60min内完成,与Real-time RT-PCR和常规RT-PCR相比,分别节省了近30 min和90 min。本方法检测极限是81拷贝/管,与Real-time RT-PCR比较,敏感性相同,均比普通RT-PCR方法高10倍。本法有较高的特异性和重复性,未发现假阳性结果。临床标本的检测结果与Real-time RT-PCR检测结果具有良好的一致性。
结论：
1.2005年～2009年,深圳市HFMD患者的CA16检出率为21.4%,CA16是该期间HFMD的主要病原之一；CA16主要感染5岁以下儿童,男女性别之间发病没有显著性差异；发病高峰为每年的夏秋季节,病例大多集中在3-6月,9-11月还有一次小流行；郊区CA16感染显著高于城区。
2.深圳市检测到的CA16可分为A、B两种基因型,B型可分为B1、B2亚型,B2亚型又可分为B2a、B2b、B2c基因亚群,之前报道的B和C基因型应归于同一B基因型下二个分支,B基因型应该是B1基因亚型,C基因型应该是B2基因亚型。各基因型间氨基酸变异位点分析未发现特异性突变,说明CA16的进化速度缓慢；69株深圳分离株基因型别为B2a和B2b,其中B2a有46株,占66.7%,B2b有23株,占33.3%；69株CA16深圳分离株VP1区核苷酸和氨基酸同源性均较高,且每年分离株组内核苷酸和氨基酸同源性均较高。
3.首次针对CA16的VPl基因设计RT-LAMP引物,建立了RT-LAMP方法,并对其特异性和敏感性进行了验证。
4.本研究建立的RT-LAMP方法在反应时间方面,比RT-PCR快近90 min,比Real-time RT-PCR快30 min；在单样本检测成本方面,比Real-time RT-PCR低约4倍,比RT-PCR低2倍。
5.本研究建立的RT-LAMP方法对临床诊断HFMD患者标本进行CA16基因检测结果与Real-time RT-PCR结果具有良好的一致性。
Background and objective
Hand, foot, and mouth disease (HFMD) is a common infectious disease of young children associated with infection of Enteroviruses, this disease has high infections and complex transmission route which could course large outbreaks in a short time. Coxsackievirus A16 (CA16) and human Enterovirus 71 (HEV71) are major causative agents of this disease. HFMD is typically mild and self-limiting, followed by developing vesicular lesions on hands, feet, mouths, and buttocks, severe cases usually presented with neurologic complications including death. EV71-associated HFMD are more severe than the disease caused by CA16 so that little attention was paid to CA16 strains, few studies was fully described CA16 molecular characteristics and no unified standard was used to divide genotype. CA16 possibly has a higher percentage than it actually is considering it's mild during the epidemic of HFMD. In recent years, fatal CA16 infection has also been described in children who had HFMD associated with myocarditis and in an adult with pneumonitis. Therefore, the epidemiological investigation and gene characteristics of CA16 should be determined. This study was conducted to investigate the casual causes of CA16 in HFMD patients during five-year study in Shenzhen, China from 2005 to 2009. To sequence the VP1 gene of CA16 isolated, it'll be analyzed the nucleotide identity with other strains at home and abroad. At the same time it will provide information on continuously monitor epidemiological and molecular evolution of CA16, and a high degree of vigilance should be maintained over the disease situation.
There are many conventional methods to detect CA16 in HFMD, such as virus isolation and cultivation, immunohistochemistry, neutralization test and reverse transcription-polymerase chain reaction (RT-PCR) etc. Classic cell separation cultivation techniques and specific antibody test method have cumbersome steps and long cycle, and some enteroviruses are difficult to cultivate in the cell lines, which easilly lead to some viruses are missed. Molecular biology diagnostic techniques based on the nucleic acid amplification such as PCR, RT-PCR and real-time fluorescent RT-PCR play a significant role in detection of pathogenic micro-organisms and disease diagnosis. Although these techniques are highly sensitive, they are not suitable for evaluation in field situation due to the involvement of complex procedures, sophisticated and expensive laboratory equipments, numerous trained workers and the application of toxic reagents such as ethidium bromide. Therefore, this developed RT-LAMP technology can provide rapid and sensitive diagnosis of CA16, which can be ideally applied in situ without transferring samples to central laboratory.
Loop-mediated isothermal amplification (LAMP) was established as a new nucleic acid amplification method by Notomi in 2000, This method used Bst DNA polymerase large fragment and four or six specially designed primers that target six or eight independent regions on DNA sequence to accomplish auto-cycling strand displacement DNA synthesis. Reaction results can be amplified by product of the precipitation of magnesium pyrophosphate to determine turbidity, also can be added SYBR Green I, ethidium bromide (EB) or other nucleic acid dyes for coloring to watch. The advantage is specificity, amplification efficiency, responsive and easy to operate. The technology has been applied to a variety of bacterial and viral gene detection by domestic and foreign scholars because of its specificity, amplification efficiency, responsive and easy to operate. The report about the technology used in detection of CA16 gene has not yet retrieved. In this paper, two pairs of specific primers were designed to capsid protein VP1 gene sequence of CA16. RT-LAMP amplification system was optimized for the detection of the target sequences, and its specificity and sensitivity were verified through experiments. The examinations have carried out to detect CA16 gene in the specimens of clinical diagnosis HFMD Patient through this technology in Shenzhen city.The result of RT-LAMP was compared with the result of reverse transcription-Polymerase chain reaction (RT-PCR) and fluorescence quantitative RT-PCR (Real-time RT-PCR) methods. The purpose is to seek fast, simple and practical new methods that applied for clinical diagnosis of CA16 infection.
Methods
1. Molecular epidemiology characteristics of CA16
CA16 positive strains were diagnosed by real-time RT-PCR to conduct further genetic analysis. A total of 69 CA16 were selected to amplify the whole VP1 region by RT-PCR techniques, using specific primers. A phylogenetic tree was constructed by comparison of the sequences with all subgenotypes of CA16 using DNASTAR and Mega 4.1 software.
2. Establishment of RT-LAMP for detection CA16
VP1 nucleotide sequences were useful for describing different genotypes of CA16 strains. The complete CA16 VP1 sequences of all genotypes were available in the Gene Bank database and aligned using CLUST X software to identify conserved regions. The VP1 sequence were analyzed by RT-LAMP primer design software and several sets of four specific primers were automatically designed. To optimize the reaction condition of RT-LAMP, and then to confirm sensitivity, specificity and repeatability of optimization RT-LAMP. Finally, evaluate RT-LAMP assay using clinical samples.
Results
1. Analysis molecular epidemiology characteristics of CA16
A total of 1,161 cases of HFMD were collected by the Center for Disease Control and Prevention in Shenzhen from 2005 to 2009, CA16 was detected in the samples collected in each year, and total positive rate of CA16 strains during real-time RT-PCR was 21.4%. The annual detection rates were 27.0%,51.6%,27.6%, 17.8%and 17.0%, respectively. The data revealed that a majority of these patients (92.7%) were less than 5 years old, no statistically significant difference was observed between boys and girls in total incidence rate of CA16 infection in the period of five years (P=0.469). Overall, monthly detection suggested that two peaks were associated with CA16 infection. The first peak occurred from March to June and the second peak was evident from September to November. Meanwhile, the first peak was consistently higher than the second. Most patients were concentrated in Longgang and Baoan and less cases in Futian, Nanshan, Luohu and Yantian.
The mean nucleotide and amino acid homology of 69 CA16 isolated from Shenzhen were 96.8% and 99.9%, respectively. In order to further determine the molecular epidemiology and genotype of Shenzhen CA16 strains, a phylogenetic dendrogram was constructed with 25 CA16 strains collected from Shenzhen and 37 genotype-identified CA16 sequences from other Chinese locations or other countries, according to similar genotypes standard of EV71, a difference of at least 15% in the VP1 gene was used to distinguish genotypes, CA16 strains were divide into two different major clusters, genotypes A and B, genotype B could be chronologically divided into B1 and B2 subtypes, and subtype B2 could be further divided into B2a, B2b and B2c. Genotype B and C in previous studies should be combined into genotype B. Genotype B in their study was considered as B1 Subtype, and genotype B was considered as B2 in our study. Amino acid evolution in the VP1 region was small, even though higher activity on the same subtypes than different ones was exhibited. All CA16 strains detected in this study belonged to cluster B2a (66.7%)and B2b (33.3%). Thus, recent genotypes of CA16 in Shenzhen were similar to those detected in other Chinese provinces and countries.
2. RT-LAMP for detection CA16
The CA16 RT-LAMP assay is fast, simple and low cost, the reaction could be finished in sixty minutes, which was less than that of conventional RT-PCR (90 min) and Real-time RT-PCR (20 min). The detection limit of this assay was 81 copies/tube, which was ten-fold higher in sensitivity than RT-PCR and equal to real-time RT-PCR. This assay aslo has high specificity and repeatability, and on false positive was found. A good correlation between RT-LAMP and real-time RT-PCR was observed on the basis of the analysis of clinical samples.
Conclutions
1. Total positive rate of CA16 strains was 21.4% from 2005 to 2009. CA16 is one of the main causative agents of HFMD in Shenzhen, high-risk group was children less than five years old, March to June and September to November are the epidemic season of this disease, suburban children are relatively more susceptibility to CA16-associated HFMD than urban children, no statistically significant difference was observed between boys and girls in incidence rate of CA16.
2. CA16 strains were divide into genotypes A and B, genotype B could be divided into subtype B1 and subtype B2, and subtype B2 could be further divided into B2a, B2b and B2c.The homogeneity of nucleotide sequence and amino acid of CA16 strains circulating in those five years high in study. All CA16 belong to cluster B2a, also there was cluster B2b.
3. In this paper, specific RT-LAMP primers were designed according to the conserved region of VP1 genome for the first time. A one-step, single-tube RT-LAMP assay was developed and validated for the detection of CA16.
4. RT-LAMP method in this assay is 90 minutes faster than RT-PCR and 30 minutes faster than Real-time RT-PCR in reaction time, and is quadruple less than RT-PCR and twice less than Real-time RT-PCR in the test cost.
5. A good correlation between the RT-LAMP and real-time PCR results was observed during the evaluation of clinical samples which were diagnosed as HFMD.
儿童手足口病(hand-foot-mouth disease, HFMD)是一种常见的传染性强、传播途径复杂、在短时间内可造成较大规模流行病毒感染性疾病,其主要病原体是柯萨奇病毒A组16型(Coxsackievirus A16, CA16)和肠道病毒71型(Enterovirus71,EV71)。大多数HFMD患者症状轻微,主要以手,足、口等部位皮肤粘膜的疱疹、溃疡为主要特征,伴有全身症状如发热、厌食、乏力、精神萎靡等。少数患儿可出现中枢神经、呼吸系统损害,引发脑炎、急性弛缓性麻痹、脑水肿、肺水肿和心肌炎等,个别重症患儿病情进展快,易发生死亡。EV71由于能引起严重的神经系统相关症状而得到高度重视,对其研究也较为深入。相反,CA16却由于引起较轻的临床症状不受关注,一直被作为需要与EV71鉴别诊断的病毒附带进行研究,流行病学资料很少,也没有统一的基因分型标准。然而,近年已有报道指出CA16感染可能与心肌炎、心包炎及其它严重疾病有关,甚至还可引起成人致死性肺炎。因此,开展CA16感染的流行病学研究,可为防控CA16病毒引起的相关疾病提供基础信息。基于此,本研究拟对2005-2009年期间深圳市的HFMD患者进行CA16病原学检测,对CA16分离株VPl区进行核苷酸序列测定,并与国内外CA16毒株进行同源性分析,旨在了解深圳地区CA16病毒的流行情况及基因型特征,以期为HFMD的综合防治和CA16病毒的动态监测提供依据,并丰富CA16毒株基因库及分子流行病学资料。
CA16常规检测方法主要有病毒分离、免疫组化、中和试验及逆转录聚合酶链反应(RT-PCR).经典的细胞分离培养、特异性抗体检测方法步骤烦琐,检测周期长,而且一些肠道病毒难以在细胞中进行增殖,容易出现漏检；基于CA16核酸扩增的分子生物学诊断技术如PCR、RT-PCR、Real-Time RT-PCR (real-time fluorescent RT-PCR)等在病原微生物的检测以及疫病诊断方面发挥着重大作用,克服了以上缺点,敏感度、特异性较高,已成为目前CA16快速诊断的重要手段,但这些方法或者需要精密控温设备和高级复杂的分析仪器,或者对操作人员的熟练度和专业水平要求比较高,且反应时间长,不利于现场样品的检测,不利于在基层推广,无法满足简单、快速、准确诊断的要求,而疫病的防控和治疗的关键在于对病原微生物的快速、准确、及早的检测并确诊。因此,建立一种快速、准确的新型诊断技术对于CA16的诊断就显得尤为重要。
环介导等温扩增(Loop-mediated isothermal amplification, LAMP)技术是Notomi等2000年建立的一种新的核酸扩增方法。其原理是利用一种链置换DNA聚合酶(Bst DNA polymerase)和两对特殊的引物,特异地识别靶序列上的6个独立区域,在等温条件下(65℃左右)保温几十分钟,即可完成核酸扩增反应。反应结果可直接靠扩增副产物焦磷酸镁的沉淀浊度进行判断,也可以由添加SYBR Green I、溴化乙啶(EB)或其它核酸染剂进行呈色后观察。其优点是特异性强,扩增效率高,反应灵敏,操作简单。LAMP技术目前已被国内外学者应用到多种细菌及病毒的基因检测中,但至今尚未检索到使用该技术检测CA16的报道。本研究拟建立CA16病毒逆转录环介导等温扩增(RT-LAMP)技术,并比较该技术与RT-PCR及Real-Time RT-PCR对深圳市临床诊断HFMD患者标本的CA16基因检测效果,旨在为临床CA16感染诊断寻求快速、简便、可行的新方法。
方法
1．CA16病毒分子流行病学特征
首先用荧光RT-PCR方法对深圳地区2005-2009年5年间所有HFMD病例进行CA16病毒快速检测,再选取每年具有代表性的阳性株用CA16特异性引物对VPl基因进行RT-PCR扩增,阳性扩增产物用于核苷酸序列测定,所得的序列与CA16各基因型代表株进行比较,用DNASTAR和Mega 4.1软件进行种系进化分析。
2.CA16病毒RT-LAMP检测方法的建立
首先针对CA16VP1区保守序列设计RT-LAMP引物,对引物进行反复筛选,选择最优引物组合,其次对反应体系进行优化,再对优化的RT-LAMP体系进行灵敏性、特异性、重复性实验验证,并运用于临床样本检测,初步评价CA16 RT-LAMP检测技术的可操作性。
结果：
1．CA16病毒分子流行病学特征分析
2005年～2009年共收集HFMD患者标本1161份,CA16检出占HFMD患者的21.4%,每年的检出率分别为：2005年27.0%,2006年51.6%,2007年27.6%；2008年17.8%；2009年17.0%。主要引起5岁以下儿童(92.7%)的感染,男女性别之间发病差异无统计学意义(P=0.469),发病高峰为每年的夏秋季节,病例大多集中在3-6月,9-11月还有一次小流行,龙岗区、宝安区(郊区)的发病显著高于福田区、南山区、罗湖区、盐田区(城区)。
本研究选取的69株CA16深圳分离株VP1区核苷酸和氨基酸同源性分别为96.8%和99.9%。基于25株深圳代表株与38株国内国际参考株的比较分析,参考EV71分型标准及Perera等的CA16分型标准,CA16可分为A、B两种基因型,B型可分为B1、B2亚型,B2亚型又可分为B2a、B2b、B2c基因亚群,之前报道的B和C基因型应归于同一B基因型下二个分支,B基因型应该归于B1基因亚型,C基因型应该归于B2基因亚型。各基因型间氨基酸变异位点分析未发现特异性突变。2005-2009年深圳分离株在CA16进化树上分别位于B2a(66.7%)和B2b(33.3%)分支上,且与同一时期同一基因型别国内国际CA16分离株的同源性较高。
2.CA16病毒RT-LAMP检测方法
建立的RT-LAMP检测方法快速、简便、成本低,反应在60min内完成,与Real-time RT-PCR和常规RT-PCR相比,分别节省了近30 min和90 min。本方法检测极限是81拷贝/管,与Real-time RT-PCR比较,敏感性相同,均比普通RT-PCR方法高10倍。本法有较高的特异性和重复性,未发现假阳性结果。临床标本的检测结果与Real-time RT-PCR检测结果具有良好的一致性。
结论：
1.2005年～2009年,深圳市HFMD患者的CA16检出率为21.4%,CA16是该期间HFMD的主要病原之一；CA16主要感染5岁以下儿童,男女性别之间发病没有显著性差异；发病高峰为每年的夏秋季节,病例大多集中在3-6月,9-11月还有一次小流行；郊区CA16感染显著高于城区。
2.深圳市检测到的CA16可分为A、B两种基因型,B型可分为B1、B2亚型,B2亚型又可分为B2a、B2b、B2c基因亚群,之前报道的B和C基因型应归于同一B基因型下二个分支,B基因型应该是B1基因亚型,C基因型应该是B2基因亚型。各基因型间氨基酸变异位点分析未发现特异性突变,说明CA16的进化速度缓慢；69株深圳分离株基因型别为B2a和B2b,其中B2a有46株,占66.7%,B2b有23株,占33.3%；69株CA16深圳分离株VP1区核苷酸和氨基酸同源性均较高,且每年分离株组内核苷酸和氨基酸同源性均较高。
3.首次针对CA16的VPl基因设计RT-LAMP引物,建立了RT-LAMP方法,并对其特异性和敏感性进行了验证。
4.本研究建立的RT-LAMP方法在反应时间方面,比RT-PCR快近90 min,比Real-time RT-PCR快30 min；在单样本检测成本方面,比Real-time RT-PCR低约4倍,比RT-PCR低2倍。
5.本研究建立的RT-LAMP方法对临床诊断HFMD患者标本进行CA16基因检测结果与Real-time RT-PCR结果具有良好的一致性。
Background and objective
Hand, foot, and mouth disease (HFMD) is a common infectious disease of young children associated with infection of Enteroviruses, this disease has high infections and complex transmission route which could course large outbreaks in a short time. Coxsackievirus A16 (CA16) and human Enterovirus 71 (HEV71) are major causative agents of this disease. HFMD is typically mild and self-limiting, followed by developing vesicular lesions on hands, feet, mouths, and buttocks, severe cases usually presented with neurologic complications including death. EV71-associated HFMD are more severe than the disease caused by CA16 so that little attention was paid to CA16 strains, few studies was fully described CA16 molecular characteristics and no unified standard was used to divide genotype. CA16 possibly has a higher percentage than it actually is considering it's mild during the epidemic of HFMD. In recent years, fatal CA16 infection has also been described in children who had HFMD associated with myocarditis and in an adult with pneumonitis. Therefore, the epidemiological investigation and gene characteristics of CA16 should be determined. This study was conducted to investigate the casual causes of CA16 in HFMD patients during five-year study in Shenzhen, China from 2005 to 2009. To sequence the VP1 gene of CA16 isolated, it'll be analyzed the nucleotide identity with other strains at home and abroad. At the same time it will provide information on continuously monitor epidemiological and molecular evolution of CA16, and a high degree of vigilance should be maintained over the disease situation.
There are many conventional methods to detect CA16 in HFMD, such as virus isolation and cultivation, immunohistochemistry, neutralization test and reverse transcription-polymerase chain reaction (RT-PCR) etc. Classic cell separation cultivation techniques and specific antibody test method have cumbersome steps and long cycle, and some enteroviruses are difficult to cultivate in the cell lines, which easilly lead to some viruses are missed. Molecular biology diagnostic techniques based on the nucleic acid amplification such as PCR, RT-PCR and real-time fluorescent RT-PCR play a significant role in detection of pathogenic micro-organisms and disease diagnosis. Although these techniques are highly sensitive, they are not suitable for evaluation in field situation due to the involvement of complex procedures, sophisticated and expensive laboratory equipments, numerous trained workers and the application of toxic reagents such as ethidium bromide. Therefore, this developed RT-LAMP technology can provide rapid and sensitive diagnosis of CA16, which can be ideally applied in situ without transferring samples to central laboratory.
Loop-mediated isothermal amplification (LAMP) was established as a new nucleic acid amplification method by Notomi in 2000, This method used Bst DNA polymerase large fragment and four or six specially designed primers that target six or eight independent regions on DNA sequence to accomplish auto-cycling strand displacement DNA synthesis. Reaction results can be amplified by product of the precipitation of magnesium pyrophosphate to determine turbidity, also can be added SYBR Green I, ethidium bromide (EB) or other nucleic acid dyes for coloring to watch. The advantage is specificity, amplification efficiency, responsive and easy to operate. The technology has been applied to a variety of bacterial and viral gene detection by domestic and foreign scholars because of its specificity, amplification efficiency, responsive and easy to operate. The report about the technology used in detection of CA16 gene has not yet retrieved. In this paper, two pairs of specific primers were designed to capsid protein VP1 gene sequence of CA16. RT-LAMP amplification system was optimized for the detection of the target sequences, and its specificity and sensitivity were verified through experiments. The examinations have carried out to detect CA16 gene in the specimens of clinical diagnosis HFMD Patient through this technology in Shenzhen city.The result of RT-LAMP was compared with the result of reverse transcription-Polymerase chain reaction (RT-PCR) and fluorescence quantitative RT-PCR (Real-time RT-PCR) methods. The purpose is to seek fast, simple and practical new methods that applied for clinical diagnosis of CA16 infection.
Methods
1. Molecular epidemiology characteristics of CA16
CA16 positive strains were diagnosed by real-time RT-PCR to conduct further genetic analysis. A total of 69 CA16 were selected to amplify the whole VP1 region by RT-PCR techniques, using specific primers. A phylogenetic tree was constructed by comparison of the sequences with all subgenotypes of CA16 using DNASTAR and Mega 4.1 software.
2. Establishment of RT-LAMP for detection CA16
VP1 nucleotide sequences were useful for describing different genotypes of CA16 strains. The complete CA16 VP1 sequences of all genotypes were available in the Gene Bank database and aligned using CLUST X software to identify conserved regions. The VP1 sequence were analyzed by RT-LAMP primer design software and several sets of four specific primers were automatically designed. To optimize the reaction condition of RT-LAMP, and then to confirm sensitivity, specificity and repeatability of optimization RT-LAMP. Finally, evaluate RT-LAMP assay using clinical samples.
Results
1. Analysis molecular epidemiology characteristics of CA16
A total of 1,161 cases of HFMD were collected by the Center for Disease Control and Prevention in Shenzhen from 2005 to 2009, CA16 was detected in the samples collected in each year, and total positive rate of CA16 strains during real-time RT-PCR was 21.4%. The annual detection rates were 27.0%,51.6%,27.6%, 17.8%and 17.0%, respectively. The data revealed that a majority of these patients (92.7%) were less than 5 years old, no statistically significant difference was observed between boys and girls in total incidence rate of CA16 infection in the period of five years (P=0.469). Overall, monthly detection suggested that two peaks were associated with CA16 infection. The first peak occurred from March to June and the second peak was evident from September to November. Meanwhile, the first peak was consistently higher than the second. Most patients were concentrated in Longgang and Baoan and less cases in Futian, Nanshan, Luohu and Yantian.
The mean nucleotide and amino acid homology of 69 CA16 isolated from Shenzhen were 96.8% and 99.9%, respectively. In order to further determine the molecular epidemiology and genotype of Shenzhen CA16 strains, a phylogenetic dendrogram was constructed with 25 CA16 strains collected from Shenzhen and 37 genotype-identified CA16 sequences from other Chinese locations or other countries, according to similar genotypes standard of EV71, a difference of at least 15% in the VP1 gene was used to distinguish genotypes, CA16 strains were divide into two different major clusters, genotypes A and B, genotype B could be chronologically divided into B1 and B2 subtypes, and subtype B2 could be further divided into B2a, B2b and B2c. Genotype B and C in previous studies should be combined into genotype B. Genotype B in their study was considered as B1 Subtype, and genotype B was considered as B2 in our study. Amino acid evolution in the VP1 region was small, even though higher activity on the same subtypes than different ones was exhibited. All CA16 strains detected in this study belonged to cluster B2a (66.7%)and B2b (33.3%). Thus, recent genotypes of CA16 in Shenzhen were similar to those detected in other Chinese provinces and countries.
2. RT-LAMP for detection CA16
The CA16 RT-LAMP assay is fast, simple and low cost, the reaction could be finished in sixty minutes, which was less than that of conventional RT-PCR (90 min) and Real-time RT-PCR (20 min). The detection limit of this assay was 81 copies/tube, which was ten-fold higher in sensitivity than RT-PCR and equal to real-time RT-PCR. This assay aslo has high specificity and repeatability, and on false positive was found. A good correlation between RT-LAMP and real-time RT-PCR was observed on the basis of the analysis of clinical samples.
Conclutions
1. Total positive rate of CA16 strains was 21.4% from 2005 to 2009. CA16 is one of the main causative agents of HFMD in Shenzhen, high-risk group was children less than five years old, March to June and September to November are the epidemic season of this disease, suburban children are relatively more susceptibility to CA16-associated HFMD than urban children, no statistically significant difference was observed between boys and girls in incidence rate of CA16.
2. CA16 strains were divide into genotypes A and B, genotype B could be divided into subtype B1 and subtype B2, and subtype B2 could be further divided into B2a, B2b and B2c.The homogeneity of nucleotide sequence and amino acid of CA16 strains circulating in those five years high in study. All CA16 belong to cluster B2a, also there was cluster B2b.
3. In this paper, specific RT-LAMP primers were designed according to the conserved region of VP1 genome for the first time. A one-step, single-tube RT-LAMP assay was developed and validated for the detection of CA16.
4. RT-LAMP method in this assay is 90 minutes faster than RT-PCR and 30 minutes faster than Real-time RT-PCR in reaction time, and is quadruple less than RT-PCR and twice less than Real-time RT-PCR in the test cost.
5. A good correlation between the RT-LAMP and real-time PCR results was observed during the evaluation of clinical samples which were diagnosed as HFMD.
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