新型多重荧光探针法检测常见肺炎链球菌血清型的临床应用研究
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摘要
肺炎链球菌(Streptococcus pneumoniae, SP)是人类最早认识的革兰阳性双球菌,从新生儿至儿童期SP的定植率可达30～100%,导致人类各种肺炎链球菌相关性疾病(Pneumococcal disease, PD):如全身严重侵袭性感染,以及局部粘膜感染,是全球范围内最常见和首要的导致社区获得性肺炎(Community-acquired pneumonia, CAP)的病原体。
儿童和长者、慢性病、酗酒、免疫力低下者易出现SP的严重感染和高病死率,PD是5岁以下儿童的首要死因,全球每年约100万婴幼儿死于该病,其中发展中国家受累最重,非洲、亚洲儿童PD的死亡率占全球的95%,我国PD死亡儿童人数列全球前10名以内,由于人口流动、多重耐药性传播等因素,全球PD负担尚在不断加重,防治压力巨大。
SP强大的致病和定植能力与其实际存在超过90种的血清型(serotype)/变种(variant)有关,决定血清型特异性的是SP细胞外包绕的荚膜多糖(Capsular polysaccharide, CPS),是SP最重要的毒力因子和致病的关键,其可作为宿主抗体的作用靶点,这也是疫苗应用的基础。迄今,根据SP的不同CPS结构,采用免疫学方法已经鉴定出46个血清群(serogroup),93个血清型。
SP的血清型与其致病性、耐药性和疫苗有效性等均密切相关,是防治PD相关研究的核心。全球范围内,13种血清型导致了超过75%的儿童侵袭性PD(Invasive pneumococcal disease, IPD);多项研究显示SP多重耐药菌(Multidrug resistance, MDR)在全球广泛流行,亚洲国家最显著,我国MDRSP高达80～90%以上,由于选择压力,流行血清型更具有耐药性,为更好的控制耐药流行SP的传播,目前已有多种针对不同血清型的疫苗,可供2岁以下儿童使用的7价结合疫苗(Pneumococcal conj ugate vaccine, PCV)全球应用最早最广。应用PCV7之初,显著降低了IPD的发病率及耐药血清型的流行,随后全球各地均出现不同程度的SP非疫苗血清型(Nonvaccine serotype, NVT)增加,总体PD的发病率不再显著降低,疫苗的有效性和研发新型疫苗是目前国际相关研究关注的焦点。
SP的血清型分布流行特点尚具有显著的地域、年龄差别,并随时间、环境变迁、人群流动、抗菌素压力、细菌进化和重组等多种因素出现不同时期流行血清型的飘移(Serotype swift),因此,持续监测SP的血清型流行分布及改变特征,与研究SP的致病机制、耐药性传播、血清型置换等实验和临床研究都息息相关,对防治PD具有重要的流行病学意义。
目前检测SP血清型的金标准是通过126种CPS兔抗血清,采用棋盘筛选方式,将SP进行血清分群和分型(可以区分91种血清型),但检测费用极昂贵,并且手工操作复杂、费时,结果判断主观,均限制其在大部分临床实验室的应用,不能适应以发展中国家和地区为首的对血清型检测的广泛需求。
随着SP的93种血清型的cps序列的公布,各种分子分型技术有望替代传统方法,针对cps序列不同靶基因的多种聚合酶链反应(Polymerase Chain Reaction,PCR)再辅助后续的不同产物分离以区分不同血清型的分子方法不断被探索。其中,美国CDC推荐采用的多重PCR (mutiple-PCR, mPCR)检测SP血清型的方案较为经济、快速、灵活和有效,目前得到全球多个研究者的采用,该方案最大的缺点是一次mPCR包括的引物对仅2-4对,需要7-8次连续mPCR才能鉴定出多种血清型,且通过电泳分析产物容易造成污染。
因此,本研究拟建立并完善高通量、高特异性且简易经济、可靠客观的检测SP血清型体系使其能广泛应用于临床实验室。
多重连接酶依赖的探针扩增法(Multiplex Ligation-dependent Probe Amp-lification, MLPA)是一种针对多种待检DNA序列进行定性和半定量分析的基于特异性探针连接的PCR新技术,基本原理包括探针和靶序列DNA进行序列特异性杂交,连接酶连接、通用引物对所有完成连接的序列进行PCR扩增,产物分离及数据分析,其特点是在一次反应中通过多重特异性探针可检测40-50个核苷酸序列,连接酶保证反应的特异性,且后续只需要一个通用引物即可进行PCR扩增,具有高通量、高特异性和简易经济的特点,目前已经应用于多个领域、多种疾病的研究,包括染色体非整倍体改变,单核苷酸多态性(Single Nucleotide Polymorphism, SNP)和点突变,多种呼吸道病毒的检测,尚未见报道用于细菌血清型分型检测。
MLPA的技术难点在于设计特异性杂交探针,模板DNA必须与两条特异性探针(长探针和短探针)均完全互补才能进行后续的连接和PCR扩增,因此可检测出一个碱基的差异,理论上很适于检测SP不同血清型cps间具有的异质性差异,MLPA后续产物的分析通常采用毛细管凝胶电泳,根据产物的大小区别,检测成本较高,耗时并有开盖产物污染风险。
本课题组对MLPA探针及后续产物分析进行改进,通过设计新型的基于与MLPA长探针上的填充序列相同的具有特定Tm的值荧光检测探针加入体系以标记不同MLPA探针反义链,在PCR扩增完成后,通过不同的荧光检测探针的融解曲线分析检测是否有相应血清型扩增产物,实现单管不开盖检测产物,极大的优化实验步骤,使其更适于临床菌株血清型检测。
本研究结合PCV7疫苗血清型和国内已有流行血清型分布研究结果,建立并完成优化常见的十种血清型的新型多重荧光探针法检测体系,一次完成对血清型4、6、9V/9A、14、15A/F、15B/C、18(18A/18B/18C/18F、19A、19F、23F的检测,并对其进行临床菌株应用评价。
第一章建立新型多重荧光探针法检测肺炎链球菌血清型体系
首先,参考美国CDC网站(http://www.cdc.gv/ncidod/biotech/strep/pcr.htm)公布信息,构建了十种血清型特异性PCR检测体系和3个mPCR检测体系,并构建完成十种血清型特异性质粒用于后续实验标准品；通过检测二十种已知血清型、SP血清型特异性质粒完善和优化检测体系,以完成对十种血清型的PCR对照检测,使其可以应用于临床菌株血清型对照检测。
其次,建立新型多重荧光探针法检测十种血清型体系：设计十种血清型特异性MLPA探针、十种血清型特异性荧光检测探针,两种内标MLPA探针及荧光检测探针。血清型特异性MLPA探针由左探针和右探针组成,左探针包括一段通用引物结合序列和一段血清型特异性寡核苷酸(LPO),右探针包括一段血清型特异性寡核苷酸(RPO)、一段填充序列(该序列与荧光检测探针序列相同)和一段通用引物结合序列。LPO和RPO序列部分的Tm值为70度左右,RPO的5’端磷酸化。当LPO和RPO杂交序列与待检测DNA特异性互补杂交,经连接酶连接后,左右两探针相连成为一个完整模板DNA,在通用引物的作用下,经不对称PCR扩增出大量产物DNA和与模板DNA互补的单链DNA。该杂交序列是参考美国CDC网站(http://www.cdc.gv/ncidod/biotech/strep/pcr.htm)公布的特异性血清型引物序列,再通过BLAST获取扩增产物序列,在产物序列里面筛选Tm值恰当,无SNP位点为杂交序列。填充序列为与链球菌基因组无特异性结合的序列,不同填充序列与荧光探针对应,其Tm为特定值,用于区分不同扩增产物。通用引物结合序列参考文献确定,填充序列参考文献根据(G+C)的Tm值确定。血清型特异性荧光检测探针序列与填充序列相同,其3’-最后一个碱基标有ROX/CY5荧光基团。
全体系中只有1对通用引物,设计为不对称PCR,检测体系中还包括杂交缓冲液、连接PCR体系。整个体系实验流程包括杂交探针与靶DNA杂交,连接酶将已杂交的LPO和RPO连接,PCR完成对整个MLPA探针序列进行扩增,荧光融解曲线分析血清型荧光检测探针完成对血清型特异性产物的检测。
再次,完成优化新型多重荧光探针检测体系并评估其灵敏度、特异性和重复性：应用SP血清型特异性质粒、二十种已知血清型(包括十种检测体系内血清型和十种检测体系外血清型)SP菌株及多种其它非SP菌株DNA作为检测标准,对整个体系及实验流程的进行优化,优化完成体系的检测灵敏度为103copies/ml,确定荧光探针融解曲线峰值>0.1为阳性血清型特异性峰；可正确分辨出所有十种已知血清型,每个已知血清型检测结果均具有3个阳性峰：血清型特异性峰,两个内标峰；采用十种检测体系外血清型和其它非SP菌株DNA进行特异性检测,未发现血清型特异性检测峰及内标峰,特异性良好,4个浓度重复检测十种已知血清型菌株DNA显示均能检测出相同阳性结果,重复性良好。
本研究建立的新型多重荧光探针法检测常见SP血清型体系符合临床检测SP血清型的简易、高效、经济、高通量的要求,实现不开盖单管完成对十种血清型检测,初步估计应用该体系检测一株SP的血清型造价是采用传统金标准方法检测的十分之一以上,检测时间约3小时,有很好的临床应用前景。
第二章新型多重荧光探针法检测肺炎链球菌血清型的临床应用评价
本研究对临床收集的侵袭性SP菌株30例、呼吸道SP菌株180例分别进行4种方法(传统方法、新型多重荧光探针法、mPCR法、血清型特异性PCR+测序法)和3种方法(新型多重荧光探针法、mPCR法、血清型特异性PCR+测序法)检测十种血清型,以评估新型多重荧光探针法检测SP菌株血清型的临床应用价值。
30例侵袭性SP菌株血清型检测结果显示,新型多重荧光探针法一次可以检测出全部菌株血清型共6种：9例19F(30%)、7例23F(23.3%)、7例14型(23.3%)、3例6型(10%)、2例15B(6.7%)、2例19A(6.7%),一份样本只检出一种血清型,检测敏感性为100%,每种血清型的检测特异性为100%,结果与其他三种方法完全一致,完成对所有菌株血清型的检测需进行3次mPCR；
180例呼吸道SP菌株血清型检测结果显示,新型多重荧光探针法一次可以检测出93.3%菌株的血清型(168/180),其中检出19F最多,占29.4%(53),其次为：23F16.1%(29)、6型15%(27)19A11.7%(21),15B5.6%(10)、14型3.3%(4),15A、18C、9V、4各占0.6%(1),检出两种血清型混合感染占10%(18),未检出相关血清型6.7%(12),新型多重荧光探针法与金标准血清型特异性PCR+测序方法检测结果无显著性差异,一致性强(Kappa=0.845, P=0.000),与mPCR方法检测结果无显著性差异,一致性强(Kappa=0.863, P=0.000);新型多重荧光探针法和mPCR法检测呼吸道SP菌株十种血清型的准确性相同,敏感性97.6%、特异性100%、阳性预测值100%、阴性预测值75%。新型多重荧光探针法检测呼吸道菌株单种血清型感染的敏感性、特异性均为100%,mPCR法的检测敏感性、特异性为98.6%、100%。对22例具有两种血清型混合感染菌株分析显示,以6/19F混合感染最多(12),23F可与多种血清型混合感染(共4种),新型多重荧光探针法和mPCR方法对混合感染的菌株血清型检测敏感性均为81.8%(18/22),特异性均为100%(158/158),阳性预测值均为100%(18/18),阴性预测值新型多重荧光探针法、mPCR法为97.6%(158/162)、98.8%(160/162)；新型多重荧光探针法与mPCR方法检测混合血清型的一致性好(Kappa=0.941, P=0.000),两方法无显著性差异(McNemar P=0.5);新型多重荧光探针法与金标准法检测混合血清型的一致性好(Kappa=0.888, P=0.000),两方法无显著性差异(McNemar P=0.125)。经分别比较侵袭性和呼吸道SP菌株检出血清型6、14、19F、23F、15B、19A的阳性率,血清型14在侵袭性菌株中的阳性率23.3%显著高于呼吸道菌株中的阳性率3.3%,差异有显著性(x2=14.435, v=1,P=0.001),其他血清型在两种样本中分布未见显著性差异。不包括检出混合血清型,三组mPCR方法每一组mPCR分别检出侵袭性SP菌株血清型覆盖率为36.7%、46.7%、16.7%,三组mPCR方法每一组mPCR分别可检出呼吸道SP菌株血清型覆盖率为39.4%、19.4%、21.1%。
4种检测血清型方法中,本研究新型多重荧光探针法平均花费约10元／例,远低于传统方法167元／例,检测十种血清型完成时间最快3小时,远比测序法3天时间短,只需要一次PCR,采用荧光定量PCR仪直接观察结果,无污染、高通量,操作最简单。检测临床菌株的准确性与mPCR法一致,整个体系稳定,适用于临床大量SP菌株的血清型初筛检测。
第三章深圳市宝安区临床SP菌株检出血清型分布特点与疫苗覆盖率
本研究采用血清型1/3/5/7F特异性PCR补充检测第二章中的未知血清型菌株,对第二章中的临床SP菌株检出血清型进行分布特点与疫苗覆盖率的流行病学分析,结果显示：
首先,侵袭性菌株和呼吸道菌株检出血清型的PCV7/13疫苗覆盖率较高,分别为86.7%/93.3%和74.4%/86.1%(细分两种混合感染血清型结果后),不同样本类型的疫苗覆盖率之间没有显著性差异；呼吸道菌株检出两种混合感染血清型能被PCV7和PCV13覆盖的比例相同,均为81.8%,两种混合感染血清型分别表现为6+19F/6+23F/23F+19F,其余18.2%不能完全被PCV7或PCV13覆盖,如排除两种混合感染血清型结果,则侵袭性菌株的PCV7/13疫苗覆盖率均显著高于呼吸道菌株；
其次,侵袭性和呼吸道SP中非PCV7疫苗血清型分布特点：侵袭性SP中共计4例为非PCV7疫苗血清型(13.3%),其中有PCV13疫苗血清型19A2例与非PCV13疫苗血清型15B2例；呼吸道SP中共计46例为非PCV7疫苗血清型(25.6%),其中有PCV13疫苗血清型19A(43.5%)和5型(2.2%)共计45.7%；其他非PCV13疫苗血清型共计54.3%,其中与15B有关的可达26.1%(包括15B+23F2.2%,15B+19A2.2%,15B21.7%),与15A有关的占4.4%(15A+23F),与PCV7疫苗血清型23F有关的占6.7%(15A+23F4.4%,15B+23F2.2%)；与19A有关的共计45.7%(19A43.5%,15B+19A2.2%)；非PCV13未知型(除外15A/15B)23.9%。
因此,通过本研究方法对临床菌株血清型的检测,能基本阐述本地区菌株的血清型分布特点,说明本研究方法对临床菌株的血清型的检测具有重要的流行病学分析的价值。
Background
Streptococcus pneumoniae (SP) is the earliest pathogen known to man, stain with Gram-positive diplococcic. The colonization rate from newborns to children of SP is high from30to100%, Streptococcus pneumoniae can cause a variety of human-related diseases (Pneumococcal disease, PD):severe invasive infections such as sepsis and meningitis and mucosal infection. It is the most common and important cause of community-acquired pneumonia (CAP) in the worldwide.
Children and the elderly, patients with chronic disease, alcoholism, immunocompromised persons prone to serious infections and high mortality, PD is considered to be the leading cause of mortality in children under five years old in developing countries, about one million infants die annually due to the disease in the world. Child mortality with PD accounted for95%of global deaths of children within the world's top10in Africa and Asia. As population movements, the spread of multi-drug resistance and other factors, the global burden of PD is still constantly increasing, prevention pressure is huge.
SP powerful pathogenic colonization ability and their actual existence of more than90kinds of serotype (serotype)/variant (variant) is associated with the serotype-specific capsular polysaccharide (CPS) outside the SP cells. It is the most important and critical virulence factors in SP, which can serve as targets of the host antibody, which is also the basis for vaccine applications. To date, depending on the structure of CPS, using immunological methods have identified46serogroups (serogroup),93serotypes.
Serotypes are closely related to its pathogenicity, drug resistance and vaccine efficacy. It is the core of prevention PD studies. Globally,13serotypes resulted in over75%of children with invasive PD (Invasive pneumococcal disease, IPD); several studies have shown that SP multi-drug resistant (MDR) widely popular around the world, especially in Asia, MDRSP in China is up to80%to90%, due to the selection pressure, the prevalent serotypes is more resistant, to better control the spread of drug-resistant SP, there are a variety of different vaccines available in the world. The7-valent conjugate vaccine (PCV7) is available to children under2years old. It is the most popular vaccine in the world. At the beginning of the application of PCV7, the incidence of IPD and the prevalence of drug-resistant serotypes reduced significantly, however, there are different degrees of SP non-vaccine serotypes (NVT) increases around the world, the overall incidence of PD no longer significantly reduced. The vaccine effectiveness and development of new vaccines are currently the focus of international research attention.
SP serotype distribution in prevalence is still relanted to geographical, age and time, as well as the changes of a variety of environmental factors, population movements, antibiotic pressure, bacterial evolution and restructuring appear in different periods lead to prevalent serotypes drifting (Serotypes swifting). Therefore, continuous monitoring of the prevalence of SP serotypes and changes of features, experimental and clinical studies of pathogenic mechanisms of drug resistance spread, SP serotype replacement and other research is related to the prevention and treatment of PD, which has important epidemiological significance.
Currently, the gold standard for detecting SP serotypes is checkerboard screening methods using anti-rabbit serum containing126kinds of CPS, which can distinguish91kinds of serotypes. But the method is expensive, complicated, time-consuming, and the results of judgment is subjective, which can not meet the broad needs of developing countries and regions for the detection of serotype.
With the announcement of SP93cps serotypes sequence, a variety of molecular typing techniques are expected to replace the conventional method, kinds of methods are explored constantly, which based on different polymerase chain reaction (Polymerase Chain Reaction, PCR) for different target gene sequence and then assisted with cps different subsequent the product was isolated to distinguish different serotypes. Among them, mPCR detection program for SP serotype, the U.S. CDC recommended, are more economical, fast, flexible and effective, which is used by a number of researchers in this world. Whereas, the biggest drawback of the program is that a primer mPCR included only two to four pairs, so7-8consecutive mPCRs are repuired to identify a variety of serotypes, and the product analyzed by electrophoresis likely to cause pollution.
Therefore, the study aim to establish and improve a high-throughput, high specificity and economical, reliable and objective system for detection of SP serotypes, so it can be widely used in clinical laboratories.
Multiple ligase dependent probe amplification (Multiplex Ligation-dependent Probe Amplification, MLPA) is a based specific probes connected new technology for a variety of DNA sequences, which can be used to qualitative and semi-quantitative analysis. The basic principle of the method are follows:probe and the target sequence hybridization, a ligase, the ligation products are amplified by PCR, product isolated and data analysis, which can detecte40-50nucleotide sequences in one reaction by multi-specific probe, ligase ensure the specificity of the reaction, and PCR amplification can be performed by only a universal primer.This method has the advantages of high-throughput, high specificity, easy and economic, has been used in many fields and for study a variety of diseases, including changes in chromosome aneuploidy, single nucleotide polymorphisms (Single Nucleotide Polymorphism, SNP) and point mutations, detection of multiple respiratory viruses, yet there isn't any report about the detection of bacterial serotypes by this method.
The difficulty of MLPA technology is to design specific hybridization probes, the two probes can be ligated and permit PCR amplification only when two specific probes (length probe and short probe) are completely complementary to DNA template, so the change of one base can be detected. This method is adapted to detect the difference between the heterogeneity of SP different serotypes cps theoretically, MLPA ampification product analysis by capillary gel electrophoresis according to the size of amplification product, which has disadvantage of cost more, time-consuming and has risk of contamination.
Our team aim to improve the MLPA probe and analysis of the amplification product by design of new type fluorescence detection probes which have certain Tm value. The fluorescence detection probes are complementary to stuffer of MLPA long probe and can be used to label MLPA probe. The amplification product of corresponding serotype can be detected by melting curve analysis of different fluorescent detection probes after amplification is completed, Subsequently, there is no need to open lid detection products in single-tube, optimizing the experimental procedure greatly, which make it more suitable for clinical isolates serum detection.
Objective
This study, combined PCV7vaccine serotypes with the prevalent serotypes of our country, developed and optimized multiplex fluorescence probes amplification to detect ten common SP serotypes, and the detection of serotypes4,6,9V/9A,14,15A/F,15B/C,18(18A/18B/18C/18F),19A,19F,23F has been completed. The applicability of the proposed method in the clinical field was investigated by analyzing clinical isolates.
Methods and results
1. Established new multiplex fluorescence probes amplification for detection of Streptococcus pneumoniae serotype
First of all, design ten serotype-specific MLPA probes and three mPCR detection system according to http://www.cdc.gv/ncidod/biotech/strep/pcr.htm, and develop ten serotype-specific plasmids for subsequent experiment standards. Then, improve and optimize this method by detection20known SP serotypes and serotype-specific plasmid, using PCR as control of the ten serotypes, which indicate that this method can be applied in clinical strains.
Second, establish multiple fluorescent probes to detect ten kinds of serotypes: Design ten kinds of serotype-specific MLPA probes, ten serotype-specific fluorescent probes to detect the two internal standard MLPA probes and fluorescent detection probe. Each serotype-specific MLPA probe consists of one left and one right probe, the left probe contains a common primer binding sequence and serotype-specific oligonucleotide (LPO), and the right probe contains a serotype-specific oligonucleotide nucleotides (RPO), a stuffer sequence (the sequence identical to the fluorescent detection probe sequences) and a common primer. Tm of LPO and RPO is about70℃and RPO5'end is phosphorylated. When the LPO and RPO hybridized to target DNA sequences and via a ligase, the two probes connected to a complete template DNA, under the action of the common primer, amount of amplification product and single-stranded DNA complementary to template DNA can be obtained by asymmetric PCR The sequence is reference to the U.S. CDC website (http://www.cdc.gv/ncidod/biotech/strep/pcr.htm) published serotype-specific primer sequences, and then get some continuous amplification product sequences through BLAST, Screening Tm values appropriate and no SNP sequence in amplification product used as hybrid sequences. Stuffer sequence can't non-specific binding with the gene of SP, different stuffer sequence corresponding to the fluorescent probe, which has certain Tm used to distinguish the amplification product. Common primer obtained from references, Stuffer sequence is determined according to (G+C) of the Tm value. Serotype-specific fluorescence detection probe sequence is identical to stuffer sequence, and the last base of3'labeled ROX/CY5.
There is only one pair of common primer in this method, which designed for asymmetric PCR, contain a hybridization buffer, ligated PCR system. Protocol of the method is follows:probe hybridized with the target DNA, ligation of LPO and RPO by ligase, analysis of serotype-specific amplification product by fluorescent melting curve.
Thirdly, optimize this new multiplex fluorescence probes amplification and evaluate the sensitivity, specificity and repeatability:application of SP serotype-specific plasmid, twenty known serotypes (including ten kinds of serotype in this system in and ten serotype out of the system) SP strain and other non SP strain DNA as standardize, optimization of the whole system and experimental process, sensitivity is103copies/ml, determine the peak of fluorescent probe melting curve0.1 as positive serotype-specific peak; can correctly distinguish all the ten known serotypes, each known serotype have3positive peaks:serotype specific peak, two internal standard peak; specificity was performed by detection ten serotypes out of system and other non-SP strain DNA and there is no serotype-specific peak which indicate acceptable specificity. Detection4concentrations of ten known serotypes DNA display the same positive result, so the repeatability of this strategy is good.
The novel method of multiplex fluorescence probes amplification established in this study used to detect common SP serotypes has the advantages of efficient, economical, high-throughput, which can achieve the requirements of detection SP in clinic and there is no need to open the lid to finish the detection of ten serotypes. Preliminary estimated that the cost of using this method to detect a strain of SP serotype is under ten percent of traditional gold standard method, detection time is about three hours, which has good prospect for clinical application.
2. Evaluation of new multiplex fluorescence probes amplification for detection clinical SP isolates serotype
In this study,30cases invasive SP isolates and180cases of respiratory SP strains were performed four methods (traditional method, new multiplex fluorescence probe amplification, mPCR method, serotype-specific PCR and sequencing) and three kinds of methods (new multiplex fluorescence probe amplification, mPCR method, serotype-specific PCR and sequencing) to detect ten kinds of SP serotypes, which was used to evaluate the application of new multiplex fluorescence probes amplification. The result of30cases of invasive strains show that the novel method of multiple fluorescent probe amplification can detect six kinds of all serotypes:9cases19F (30%),7cases23F (23.3%),7cases14(23.3%),3cases of6(10%),2cases15B (6.7%),2cases19A (6.7%), each sample detect only one serotype, the sensitivity of is100%, specificity is100%, which were completely consistent with the other three methods, however, three mPCR was needed to detect all serotype.
The result of180cases of respiratory SP strains indicate that93.3%(168/180) of SP serotype can be detected by the new method of multiple fluorescent probe amplification, which detected19F most, accounting for29.4%(53), followed by:23F 16.1%(29),615%(27),19A11.1%(20),15B5.6%(10),143.3%(6),18C,9V,40.6%(1), two serotypes mixed infection accounted for10%(18),6.7%(12) were not detected related serotypes. The new multiplex fluorescence probe amplification and the gold standard serotype-specific PCR and sequencing have no significant differences (Kappa=0.845, P=0.000), and have no significant differences with mPCR (Kappa=0.863, P=0.000). The accuracy of novel multiplex fluorescence probe amplification is identical to mPCR for detection ten serotype, sensitivity, specificity, positive predictive value, negative predictive value were97.6%,100%,100%,75%, respectively. The sensitivity and the specificity of detection single serotype of respiratory infection strains by multiple fluorescent probe amplification were100%, mPCR were98.6%and100%, respectively. Analysis of22isolates mixed infection with two serotypes showed that major mixed infection serotype was6/19F (12), serotype23F can mixed infection with4serotype, sensitivity, specificity, positive predictive value, of novel multiplex fluorescence probe amplification and mPCR detection mixed infection were81.8%(18/22),100%(158/158),100%(18/18), negative predictive value was97.6%(158/162),98.8%(160/162), respectively. The new fluorescent probe amplification detected mixed infection consistent with mPCR (Kappa=0.941, P=0.000), two methods have no significant differences (McNemar P=0.5). The new fluorescent probe amplification detected mixed infection consistent with gold method (Kappa=0.888, P=0.000), two methods have no significant differences (McNemar P=0.125). Among isolates serotype6,14,19F,23F, the positive rate of15B,19A, and positive rate(23.3%) of invasive strains serotype14was significantly higher than respiratory isolates(3.3%), has significant differences (%2=14.435, v=1, P=0.001), distribution of other serotypes between the two samples have no significant differences. Excluding mixed infection serotypes, coverage of detection invasive serotype in each mPCR in3mPCR was36.7%,46.7%,16.7%, respectively. For respiratory SP isolates, serotype coverage was39.4%,19.4%,21.1%respectively.
3. Distribution of serotype and vaccine coverage ratio of clinical SP isolates
In this study, serotype-specific PCR supplement1/3/5/7F detect clinical SP strains,serotypes distribution and PCV vaccine coverage analysis, the results showed:
First, invasive strains and strains detected in respiratory had PCV7/13high vaccine coverage, which were86.7%/93.3%and74.4%/86.1%(after subdivision two mixed infection serotype), vaccine coverage was no significant differences between different samples type; co-infection serotypes in respiratory strain had same coverage ratio,81.8%for PCV13and PCV7, two serotypes mixed infection showed6+19F/6+23F/23F+19F, the remaining18.2%can not be fully covered by PCV7or PCV13, If exclusion of mixed infection of two serotypes result, the invasive strains PCV7/13vaccine coverage were significantly higher than respiratory isolates;
Second, invasive and respiratory SP non-PCV7vaccine serotypes distribution: invasive SP in a total of four cases non-PCV7vaccine serotypes (13.3%)of, which has PCV13vaccine serotypes19A2isolates and with non-PCV13vaccine serotypes15B2isolates; respiratory SP in a total of46cases of non-PCV7vaccine serotypes (25.6%), which has PCV13vaccine serotypes19A (43.5%) and type5(2.2%) total45.7%; other non-PCV13vaccine serotypes total of54.3%where relevant and15B up26.1%(including15B+23F2.2%,15B+19A2.2%,15B21.7%), and15A accounted for about4.4%(15A+23F), with the PCV7vaccine serotype23F accounted for about6.7%(15A+23F4.4%,15B+23F2.2%);19A associated with a total of45.7%(19A43.5%,15B+19A2.2%); non-PCV13unknown type (except15A/15B)23.9%.
Therefore, through this research method in clinical strains of serotype detected, can basically describes serotype distribution characters in this region, indicating that the value of this research method has important epidemiological significance for analysis serotype.
Conclusion
The average cost of new multiplex fluorescence probe amplification is about10yuan/case, much lower than the traditional method167yuan/case, which need least of3hours to detection ten serotypes, faster than sequencing three days, and have the advantages of only one PCR, result getting from quantitative PCR, non-polluting, high-throughput and easy to operate. The accuracy of detection clinical isolates consistent with mPCR, have good stability, which is appropriate for screening of a large number of clinical isolates of SP serotype.
儿童和长者、慢性病、酗酒、免疫力低下者易出现SP的严重感染和高病死率,PD是5岁以下儿童的首要死因,全球每年约100万婴幼儿死于该病,其中发展中国家受累最重,非洲、亚洲儿童PD的死亡率占全球的95%,我国PD死亡儿童人数列全球前10名以内,由于人口流动、多重耐药性传播等因素,全球PD负担尚在不断加重,防治压力巨大。
SP强大的致病和定植能力与其实际存在超过90种的血清型(serotype)/变种(variant)有关,决定血清型特异性的是SP细胞外包绕的荚膜多糖(Capsular polysaccharide, CPS),是SP最重要的毒力因子和致病的关键,其可作为宿主抗体的作用靶点,这也是疫苗应用的基础。迄今,根据SP的不同CPS结构,采用免疫学方法已经鉴定出46个血清群(serogroup),93个血清型。
SP的血清型与其致病性、耐药性和疫苗有效性等均密切相关,是防治PD相关研究的核心。全球范围内,13种血清型导致了超过75%的儿童侵袭性PD(Invasive pneumococcal disease, IPD);多项研究显示SP多重耐药菌(Multidrug resistance, MDR)在全球广泛流行,亚洲国家最显著,我国MDRSP高达80～90%以上,由于选择压力,流行血清型更具有耐药性,为更好的控制耐药流行SP的传播,目前已有多种针对不同血清型的疫苗,可供2岁以下儿童使用的7价结合疫苗(Pneumococcal conj ugate vaccine, PCV)全球应用最早最广。应用PCV7之初,显著降低了IPD的发病率及耐药血清型的流行,随后全球各地均出现不同程度的SP非疫苗血清型(Nonvaccine serotype, NVT)增加,总体PD的发病率不再显著降低,疫苗的有效性和研发新型疫苗是目前国际相关研究关注的焦点。
SP的血清型分布流行特点尚具有显著的地域、年龄差别,并随时间、环境变迁、人群流动、抗菌素压力、细菌进化和重组等多种因素出现不同时期流行血清型的飘移(Serotype swift),因此,持续监测SP的血清型流行分布及改变特征,与研究SP的致病机制、耐药性传播、血清型置换等实验和临床研究都息息相关,对防治PD具有重要的流行病学意义。
目前检测SP血清型的金标准是通过126种CPS兔抗血清,采用棋盘筛选方式,将SP进行血清分群和分型(可以区分91种血清型),但检测费用极昂贵,并且手工操作复杂、费时,结果判断主观,均限制其在大部分临床实验室的应用,不能适应以发展中国家和地区为首的对血清型检测的广泛需求。
随着SP的93种血清型的cps序列的公布,各种分子分型技术有望替代传统方法,针对cps序列不同靶基因的多种聚合酶链反应(Polymerase Chain Reaction,PCR)再辅助后续的不同产物分离以区分不同血清型的分子方法不断被探索。其中,美国CDC推荐采用的多重PCR (mutiple-PCR, mPCR)检测SP血清型的方案较为经济、快速、灵活和有效,目前得到全球多个研究者的采用,该方案最大的缺点是一次mPCR包括的引物对仅2-4对,需要7-8次连续mPCR才能鉴定出多种血清型,且通过电泳分析产物容易造成污染。
因此,本研究拟建立并完善高通量、高特异性且简易经济、可靠客观的检测SP血清型体系使其能广泛应用于临床实验室。
多重连接酶依赖的探针扩增法(Multiplex Ligation-dependent Probe Amp-lification, MLPA)是一种针对多种待检DNA序列进行定性和半定量分析的基于特异性探针连接的PCR新技术,基本原理包括探针和靶序列DNA进行序列特异性杂交,连接酶连接、通用引物对所有完成连接的序列进行PCR扩增,产物分离及数据分析,其特点是在一次反应中通过多重特异性探针可检测40-50个核苷酸序列,连接酶保证反应的特异性,且后续只需要一个通用引物即可进行PCR扩增,具有高通量、高特异性和简易经济的特点,目前已经应用于多个领域、多种疾病的研究,包括染色体非整倍体改变,单核苷酸多态性(Single Nucleotide Polymorphism, SNP)和点突变,多种呼吸道病毒的检测,尚未见报道用于细菌血清型分型检测。
MLPA的技术难点在于设计特异性杂交探针,模板DNA必须与两条特异性探针(长探针和短探针)均完全互补才能进行后续的连接和PCR扩增,因此可检测出一个碱基的差异,理论上很适于检测SP不同血清型cps间具有的异质性差异,MLPA后续产物的分析通常采用毛细管凝胶电泳,根据产物的大小区别,检测成本较高,耗时并有开盖产物污染风险。
本课题组对MLPA探针及后续产物分析进行改进,通过设计新型的基于与MLPA长探针上的填充序列相同的具有特定Tm的值荧光检测探针加入体系以标记不同MLPA探针反义链,在PCR扩增完成后,通过不同的荧光检测探针的融解曲线分析检测是否有相应血清型扩增产物,实现单管不开盖检测产物,极大的优化实验步骤,使其更适于临床菌株血清型检测。
本研究结合PCV7疫苗血清型和国内已有流行血清型分布研究结果,建立并完成优化常见的十种血清型的新型多重荧光探针法检测体系,一次完成对血清型4、6、9V/9A、14、15A/F、15B/C、18(18A/18B/18C/18F、19A、19F、23F的检测,并对其进行临床菌株应用评价。
第一章建立新型多重荧光探针法检测肺炎链球菌血清型体系
首先,参考美国CDC网站(http://www.cdc.gv/ncidod/biotech/strep/pcr.htm)公布信息,构建了十种血清型特异性PCR检测体系和3个mPCR检测体系,并构建完成十种血清型特异性质粒用于后续实验标准品；通过检测二十种已知血清型、SP血清型特异性质粒完善和优化检测体系,以完成对十种血清型的PCR对照检测,使其可以应用于临床菌株血清型对照检测。
其次,建立新型多重荧光探针法检测十种血清型体系：设计十种血清型特异性MLPA探针、十种血清型特异性荧光检测探针,两种内标MLPA探针及荧光检测探针。血清型特异性MLPA探针由左探针和右探针组成,左探针包括一段通用引物结合序列和一段血清型特异性寡核苷酸(LPO),右探针包括一段血清型特异性寡核苷酸(RPO)、一段填充序列(该序列与荧光检测探针序列相同)和一段通用引物结合序列。LPO和RPO序列部分的Tm值为70度左右,RPO的5’端磷酸化。当LPO和RPO杂交序列与待检测DNA特异性互补杂交,经连接酶连接后,左右两探针相连成为一个完整模板DNA,在通用引物的作用下,经不对称PCR扩增出大量产物DNA和与模板DNA互补的单链DNA。该杂交序列是参考美国CDC网站(http://www.cdc.gv/ncidod/biotech/strep/pcr.htm)公布的特异性血清型引物序列,再通过BLAST获取扩增产物序列,在产物序列里面筛选Tm值恰当,无SNP位点为杂交序列。填充序列为与链球菌基因组无特异性结合的序列,不同填充序列与荧光探针对应,其Tm为特定值,用于区分不同扩增产物。通用引物结合序列参考文献确定,填充序列参考文献根据(G+C)的Tm值确定。血清型特异性荧光检测探针序列与填充序列相同,其3’-最后一个碱基标有ROX/CY5荧光基团。
全体系中只有1对通用引物,设计为不对称PCR,检测体系中还包括杂交缓冲液、连接PCR体系。整个体系实验流程包括杂交探针与靶DNA杂交,连接酶将已杂交的LPO和RPO连接,PCR完成对整个MLPA探针序列进行扩增,荧光融解曲线分析血清型荧光检测探针完成对血清型特异性产物的检测。
再次,完成优化新型多重荧光探针检测体系并评估其灵敏度、特异性和重复性：应用SP血清型特异性质粒、二十种已知血清型(包括十种检测体系内血清型和十种检测体系外血清型)SP菌株及多种其它非SP菌株DNA作为检测标准,对整个体系及实验流程的进行优化,优化完成体系的检测灵敏度为103copies/ml,确定荧光探针融解曲线峰值>0.1为阳性血清型特异性峰；可正确分辨出所有十种已知血清型,每个已知血清型检测结果均具有3个阳性峰：血清型特异性峰,两个内标峰；采用十种检测体系外血清型和其它非SP菌株DNA进行特异性检测,未发现血清型特异性检测峰及内标峰,特异性良好,4个浓度重复检测十种已知血清型菌株DNA显示均能检测出相同阳性结果,重复性良好。
本研究建立的新型多重荧光探针法检测常见SP血清型体系符合临床检测SP血清型的简易、高效、经济、高通量的要求,实现不开盖单管完成对十种血清型检测,初步估计应用该体系检测一株SP的血清型造价是采用传统金标准方法检测的十分之一以上,检测时间约3小时,有很好的临床应用前景。
第二章新型多重荧光探针法检测肺炎链球菌血清型的临床应用评价
本研究对临床收集的侵袭性SP菌株30例、呼吸道SP菌株180例分别进行4种方法(传统方法、新型多重荧光探针法、mPCR法、血清型特异性PCR+测序法)和3种方法(新型多重荧光探针法、mPCR法、血清型特异性PCR+测序法)检测十种血清型,以评估新型多重荧光探针法检测SP菌株血清型的临床应用价值。
30例侵袭性SP菌株血清型检测结果显示,新型多重荧光探针法一次可以检测出全部菌株血清型共6种：9例19F(30%)、7例23F(23.3%)、7例14型(23.3%)、3例6型(10%)、2例15B(6.7%)、2例19A(6.7%),一份样本只检出一种血清型,检测敏感性为100%,每种血清型的检测特异性为100%,结果与其他三种方法完全一致,完成对所有菌株血清型的检测需进行3次mPCR；
180例呼吸道SP菌株血清型检测结果显示,新型多重荧光探针法一次可以检测出93.3%菌株的血清型(168/180),其中检出19F最多,占29.4%(53),其次为：23F16.1%(29)、6型15%(27)19A11.7%(21),15B5.6%(10)、14型3.3%(4),15A、18C、9V、4各占0.6%(1),检出两种血清型混合感染占10%(18),未检出相关血清型6.7%(12),新型多重荧光探针法与金标准血清型特异性PCR+测序方法检测结果无显著性差异,一致性强(Kappa=0.845, P=0.000),与mPCR方法检测结果无显著性差异,一致性强(Kappa=0.863, P=0.000);新型多重荧光探针法和mPCR法检测呼吸道SP菌株十种血清型的准确性相同,敏感性97.6%、特异性100%、阳性预测值100%、阴性预测值75%。新型多重荧光探针法检测呼吸道菌株单种血清型感染的敏感性、特异性均为100%,mPCR法的检测敏感性、特异性为98.6%、100%。对22例具有两种血清型混合感染菌株分析显示,以6/19F混合感染最多(12),23F可与多种血清型混合感染(共4种),新型多重荧光探针法和mPCR方法对混合感染的菌株血清型检测敏感性均为81.8%(18/22),特异性均为100%(158/158),阳性预测值均为100%(18/18),阴性预测值新型多重荧光探针法、mPCR法为97.6%(158/162)、98.8%(160/162)；新型多重荧光探针法与mPCR方法检测混合血清型的一致性好(Kappa=0.941, P=0.000),两方法无显著性差异(McNemar P=0.5);新型多重荧光探针法与金标准法检测混合血清型的一致性好(Kappa=0.888, P=0.000),两方法无显著性差异(McNemar P=0.125)。经分别比较侵袭性和呼吸道SP菌株检出血清型6、14、19F、23F、15B、19A的阳性率,血清型14在侵袭性菌株中的阳性率23.3%显著高于呼吸道菌株中的阳性率3.3%,差异有显著性(x2=14.435, v=1,P=0.001),其他血清型在两种样本中分布未见显著性差异。不包括检出混合血清型,三组mPCR方法每一组mPCR分别检出侵袭性SP菌株血清型覆盖率为36.7%、46.7%、16.7%,三组mPCR方法每一组mPCR分别可检出呼吸道SP菌株血清型覆盖率为39.4%、19.4%、21.1%。
4种检测血清型方法中,本研究新型多重荧光探针法平均花费约10元／例,远低于传统方法167元／例,检测十种血清型完成时间最快3小时,远比测序法3天时间短,只需要一次PCR,采用荧光定量PCR仪直接观察结果,无污染、高通量,操作最简单。检测临床菌株的准确性与mPCR法一致,整个体系稳定,适用于临床大量SP菌株的血清型初筛检测。
第三章深圳市宝安区临床SP菌株检出血清型分布特点与疫苗覆盖率
本研究采用血清型1/3/5/7F特异性PCR补充检测第二章中的未知血清型菌株,对第二章中的临床SP菌株检出血清型进行分布特点与疫苗覆盖率的流行病学分析,结果显示：
首先,侵袭性菌株和呼吸道菌株检出血清型的PCV7/13疫苗覆盖率较高,分别为86.7%/93.3%和74.4%/86.1%(细分两种混合感染血清型结果后),不同样本类型的疫苗覆盖率之间没有显著性差异；呼吸道菌株检出两种混合感染血清型能被PCV7和PCV13覆盖的比例相同,均为81.8%,两种混合感染血清型分别表现为6+19F/6+23F/23F+19F,其余18.2%不能完全被PCV7或PCV13覆盖,如排除两种混合感染血清型结果,则侵袭性菌株的PCV7/13疫苗覆盖率均显著高于呼吸道菌株；
其次,侵袭性和呼吸道SP中非PCV7疫苗血清型分布特点：侵袭性SP中共计4例为非PCV7疫苗血清型(13.3%),其中有PCV13疫苗血清型19A2例与非PCV13疫苗血清型15B2例；呼吸道SP中共计46例为非PCV7疫苗血清型(25.6%),其中有PCV13疫苗血清型19A(43.5%)和5型(2.2%)共计45.7%；其他非PCV13疫苗血清型共计54.3%,其中与15B有关的可达26.1%(包括15B+23F2.2%,15B+19A2.2%,15B21.7%),与15A有关的占4.4%(15A+23F),与PCV7疫苗血清型23F有关的占6.7%(15A+23F4.4%,15B+23F2.2%)；与19A有关的共计45.7%(19A43.5%,15B+19A2.2%)；非PCV13未知型(除外15A/15B)23.9%。
因此,通过本研究方法对临床菌株血清型的检测,能基本阐述本地区菌株的血清型分布特点,说明本研究方法对临床菌株的血清型的检测具有重要的流行病学分析的价值。
Background
Streptococcus pneumoniae (SP) is the earliest pathogen known to man, stain with Gram-positive diplococcic. The colonization rate from newborns to children of SP is high from30to100%, Streptococcus pneumoniae can cause a variety of human-related diseases (Pneumococcal disease, PD):severe invasive infections such as sepsis and meningitis and mucosal infection. It is the most common and important cause of community-acquired pneumonia (CAP) in the worldwide.
Children and the elderly, patients with chronic disease, alcoholism, immunocompromised persons prone to serious infections and high mortality, PD is considered to be the leading cause of mortality in children under five years old in developing countries, about one million infants die annually due to the disease in the world. Child mortality with PD accounted for95%of global deaths of children within the world's top10in Africa and Asia. As population movements, the spread of multi-drug resistance and other factors, the global burden of PD is still constantly increasing, prevention pressure is huge.
SP powerful pathogenic colonization ability and their actual existence of more than90kinds of serotype (serotype)/variant (variant) is associated with the serotype-specific capsular polysaccharide (CPS) outside the SP cells. It is the most important and critical virulence factors in SP, which can serve as targets of the host antibody, which is also the basis for vaccine applications. To date, depending on the structure of CPS, using immunological methods have identified46serogroups (serogroup),93serotypes.
Serotypes are closely related to its pathogenicity, drug resistance and vaccine efficacy. It is the core of prevention PD studies. Globally,13serotypes resulted in over75%of children with invasive PD (Invasive pneumococcal disease, IPD); several studies have shown that SP multi-drug resistant (MDR) widely popular around the world, especially in Asia, MDRSP in China is up to80%to90%, due to the selection pressure, the prevalent serotypes is more resistant, to better control the spread of drug-resistant SP, there are a variety of different vaccines available in the world. The7-valent conjugate vaccine (PCV7) is available to children under2years old. It is the most popular vaccine in the world. At the beginning of the application of PCV7, the incidence of IPD and the prevalence of drug-resistant serotypes reduced significantly, however, there are different degrees of SP non-vaccine serotypes (NVT) increases around the world, the overall incidence of PD no longer significantly reduced. The vaccine effectiveness and development of new vaccines are currently the focus of international research attention.
SP serotype distribution in prevalence is still relanted to geographical, age and time, as well as the changes of a variety of environmental factors, population movements, antibiotic pressure, bacterial evolution and restructuring appear in different periods lead to prevalent serotypes drifting (Serotypes swifting). Therefore, continuous monitoring of the prevalence of SP serotypes and changes of features, experimental and clinical studies of pathogenic mechanisms of drug resistance spread, SP serotype replacement and other research is related to the prevention and treatment of PD, which has important epidemiological significance.
Currently, the gold standard for detecting SP serotypes is checkerboard screening methods using anti-rabbit serum containing126kinds of CPS, which can distinguish91kinds of serotypes. But the method is expensive, complicated, time-consuming, and the results of judgment is subjective, which can not meet the broad needs of developing countries and regions for the detection of serotype.
With the announcement of SP93cps serotypes sequence, a variety of molecular typing techniques are expected to replace the conventional method, kinds of methods are explored constantly, which based on different polymerase chain reaction (Polymerase Chain Reaction, PCR) for different target gene sequence and then assisted with cps different subsequent the product was isolated to distinguish different serotypes. Among them, mPCR detection program for SP serotype, the U.S. CDC recommended, are more economical, fast, flexible and effective, which is used by a number of researchers in this world. Whereas, the biggest drawback of the program is that a primer mPCR included only two to four pairs, so7-8consecutive mPCRs are repuired to identify a variety of serotypes, and the product analyzed by electrophoresis likely to cause pollution.
Therefore, the study aim to establish and improve a high-throughput, high specificity and economical, reliable and objective system for detection of SP serotypes, so it can be widely used in clinical laboratories.
Multiple ligase dependent probe amplification (Multiplex Ligation-dependent Probe Amplification, MLPA) is a based specific probes connected new technology for a variety of DNA sequences, which can be used to qualitative and semi-quantitative analysis. The basic principle of the method are follows:probe and the target sequence hybridization, a ligase, the ligation products are amplified by PCR, product isolated and data analysis, which can detecte40-50nucleotide sequences in one reaction by multi-specific probe, ligase ensure the specificity of the reaction, and PCR amplification can be performed by only a universal primer.This method has the advantages of high-throughput, high specificity, easy and economic, has been used in many fields and for study a variety of diseases, including changes in chromosome aneuploidy, single nucleotide polymorphisms (Single Nucleotide Polymorphism, SNP) and point mutations, detection of multiple respiratory viruses, yet there isn't any report about the detection of bacterial serotypes by this method.
The difficulty of MLPA technology is to design specific hybridization probes, the two probes can be ligated and permit PCR amplification only when two specific probes (length probe and short probe) are completely complementary to DNA template, so the change of one base can be detected. This method is adapted to detect the difference between the heterogeneity of SP different serotypes cps theoretically, MLPA ampification product analysis by capillary gel electrophoresis according to the size of amplification product, which has disadvantage of cost more, time-consuming and has risk of contamination.
Our team aim to improve the MLPA probe and analysis of the amplification product by design of new type fluorescence detection probes which have certain Tm value. The fluorescence detection probes are complementary to stuffer of MLPA long probe and can be used to label MLPA probe. The amplification product of corresponding serotype can be detected by melting curve analysis of different fluorescent detection probes after amplification is completed, Subsequently, there is no need to open lid detection products in single-tube, optimizing the experimental procedure greatly, which make it more suitable for clinical isolates serum detection.
Objective
This study, combined PCV7vaccine serotypes with the prevalent serotypes of our country, developed and optimized multiplex fluorescence probes amplification to detect ten common SP serotypes, and the detection of serotypes4,6,9V/9A,14,15A/F,15B/C,18(18A/18B/18C/18F),19A,19F,23F has been completed. The applicability of the proposed method in the clinical field was investigated by analyzing clinical isolates.
Methods and results
1. Established new multiplex fluorescence probes amplification for detection of Streptococcus pneumoniae serotype
First of all, design ten serotype-specific MLPA probes and three mPCR detection system according to http://www.cdc.gv/ncidod/biotech/strep/pcr.htm, and develop ten serotype-specific plasmids for subsequent experiment standards. Then, improve and optimize this method by detection20known SP serotypes and serotype-specific plasmid, using PCR as control of the ten serotypes, which indicate that this method can be applied in clinical strains.
Second, establish multiple fluorescent probes to detect ten kinds of serotypes: Design ten kinds of serotype-specific MLPA probes, ten serotype-specific fluorescent probes to detect the two internal standard MLPA probes and fluorescent detection probe. Each serotype-specific MLPA probe consists of one left and one right probe, the left probe contains a common primer binding sequence and serotype-specific oligonucleotide (LPO), and the right probe contains a serotype-specific oligonucleotide nucleotides (RPO), a stuffer sequence (the sequence identical to the fluorescent detection probe sequences) and a common primer. Tm of LPO and RPO is about70℃and RPO5'end is phosphorylated. When the LPO and RPO hybridized to target DNA sequences and via a ligase, the two probes connected to a complete template DNA, under the action of the common primer, amount of amplification product and single-stranded DNA complementary to template DNA can be obtained by asymmetric PCR The sequence is reference to the U.S. CDC website (http://www.cdc.gv/ncidod/biotech/strep/pcr.htm) published serotype-specific primer sequences, and then get some continuous amplification product sequences through BLAST, Screening Tm values appropriate and no SNP sequence in amplification product used as hybrid sequences. Stuffer sequence can't non-specific binding with the gene of SP, different stuffer sequence corresponding to the fluorescent probe, which has certain Tm used to distinguish the amplification product. Common primer obtained from references, Stuffer sequence is determined according to (G+C) of the Tm value. Serotype-specific fluorescence detection probe sequence is identical to stuffer sequence, and the last base of3'labeled ROX/CY5.
There is only one pair of common primer in this method, which designed for asymmetric PCR, contain a hybridization buffer, ligated PCR system. Protocol of the method is follows:probe hybridized with the target DNA, ligation of LPO and RPO by ligase, analysis of serotype-specific amplification product by fluorescent melting curve.
Thirdly, optimize this new multiplex fluorescence probes amplification and evaluate the sensitivity, specificity and repeatability:application of SP serotype-specific plasmid, twenty known serotypes (including ten kinds of serotype in this system in and ten serotype out of the system) SP strain and other non SP strain DNA as standardize, optimization of the whole system and experimental process, sensitivity is103copies/ml, determine the peak of fluorescent probe melting curve0.1 as positive serotype-specific peak; can correctly distinguish all the ten known serotypes, each known serotype have3positive peaks:serotype specific peak, two internal standard peak; specificity was performed by detection ten serotypes out of system and other non-SP strain DNA and there is no serotype-specific peak which indicate acceptable specificity. Detection4concentrations of ten known serotypes DNA display the same positive result, so the repeatability of this strategy is good.
The novel method of multiplex fluorescence probes amplification established in this study used to detect common SP serotypes has the advantages of efficient, economical, high-throughput, which can achieve the requirements of detection SP in clinic and there is no need to open the lid to finish the detection of ten serotypes. Preliminary estimated that the cost of using this method to detect a strain of SP serotype is under ten percent of traditional gold standard method, detection time is about three hours, which has good prospect for clinical application.
2. Evaluation of new multiplex fluorescence probes amplification for detection clinical SP isolates serotype
In this study,30cases invasive SP isolates and180cases of respiratory SP strains were performed four methods (traditional method, new multiplex fluorescence probe amplification, mPCR method, serotype-specific PCR and sequencing) and three kinds of methods (new multiplex fluorescence probe amplification, mPCR method, serotype-specific PCR and sequencing) to detect ten kinds of SP serotypes, which was used to evaluate the application of new multiplex fluorescence probes amplification. The result of30cases of invasive strains show that the novel method of multiple fluorescent probe amplification can detect six kinds of all serotypes:9cases19F (30%),7cases23F (23.3%),7cases14(23.3%),3cases of6(10%),2cases15B (6.7%),2cases19A (6.7%), each sample detect only one serotype, the sensitivity of is100%, specificity is100%, which were completely consistent with the other three methods, however, three mPCR was needed to detect all serotype.
The result of180cases of respiratory SP strains indicate that93.3%(168/180) of SP serotype can be detected by the new method of multiple fluorescent probe amplification, which detected19F most, accounting for29.4%(53), followed by:23F 16.1%(29),615%(27),19A11.1%(20),15B5.6%(10),143.3%(6),18C,9V,40.6%(1), two serotypes mixed infection accounted for10%(18),6.7%(12) were not detected related serotypes. The new multiplex fluorescence probe amplification and the gold standard serotype-specific PCR and sequencing have no significant differences (Kappa=0.845, P=0.000), and have no significant differences with mPCR (Kappa=0.863, P=0.000). The accuracy of novel multiplex fluorescence probe amplification is identical to mPCR for detection ten serotype, sensitivity, specificity, positive predictive value, negative predictive value were97.6%,100%,100%,75%, respectively. The sensitivity and the specificity of detection single serotype of respiratory infection strains by multiple fluorescent probe amplification were100%, mPCR were98.6%and100%, respectively. Analysis of22isolates mixed infection with two serotypes showed that major mixed infection serotype was6/19F (12), serotype23F can mixed infection with4serotype, sensitivity, specificity, positive predictive value, of novel multiplex fluorescence probe amplification and mPCR detection mixed infection were81.8%(18/22),100%(158/158),100%(18/18), negative predictive value was97.6%(158/162),98.8%(160/162), respectively. The new fluorescent probe amplification detected mixed infection consistent with mPCR (Kappa=0.941, P=0.000), two methods have no significant differences (McNemar P=0.5). The new fluorescent probe amplification detected mixed infection consistent with gold method (Kappa=0.888, P=0.000), two methods have no significant differences (McNemar P=0.125). Among isolates serotype6,14,19F,23F, the positive rate of15B,19A, and positive rate(23.3%) of invasive strains serotype14was significantly higher than respiratory isolates(3.3%), has significant differences (%2=14.435, v=1, P=0.001), distribution of other serotypes between the two samples have no significant differences. Excluding mixed infection serotypes, coverage of detection invasive serotype in each mPCR in3mPCR was36.7%,46.7%,16.7%, respectively. For respiratory SP isolates, serotype coverage was39.4%,19.4%,21.1%respectively.
3. Distribution of serotype and vaccine coverage ratio of clinical SP isolates
In this study, serotype-specific PCR supplement1/3/5/7F detect clinical SP strains,serotypes distribution and PCV vaccine coverage analysis, the results showed:
First, invasive strains and strains detected in respiratory had PCV7/13high vaccine coverage, which were86.7%/93.3%and74.4%/86.1%(after subdivision two mixed infection serotype), vaccine coverage was no significant differences between different samples type; co-infection serotypes in respiratory strain had same coverage ratio,81.8%for PCV13and PCV7, two serotypes mixed infection showed6+19F/6+23F/23F+19F, the remaining18.2%can not be fully covered by PCV7or PCV13, If exclusion of mixed infection of two serotypes result, the invasive strains PCV7/13vaccine coverage were significantly higher than respiratory isolates;
Second, invasive and respiratory SP non-PCV7vaccine serotypes distribution: invasive SP in a total of four cases non-PCV7vaccine serotypes (13.3%)of, which has PCV13vaccine serotypes19A2isolates and with non-PCV13vaccine serotypes15B2isolates; respiratory SP in a total of46cases of non-PCV7vaccine serotypes (25.6%), which has PCV13vaccine serotypes19A (43.5%) and type5(2.2%) total45.7%; other non-PCV13vaccine serotypes total of54.3%where relevant and15B up26.1%(including15B+23F2.2%,15B+19A2.2%,15B21.7%), and15A accounted for about4.4%(15A+23F), with the PCV7vaccine serotype23F accounted for about6.7%(15A+23F4.4%,15B+23F2.2%);19A associated with a total of45.7%(19A43.5%,15B+19A2.2%); non-PCV13unknown type (except15A/15B)23.9%.
Therefore, through this research method in clinical strains of serotype detected, can basically describes serotype distribution characters in this region, indicating that the value of this research method has important epidemiological significance for analysis serotype.
Conclusion
The average cost of new multiplex fluorescence probe amplification is about10yuan/case, much lower than the traditional method167yuan/case, which need least of3hours to detection ten serotypes, faster than sequencing three days, and have the advantages of only one PCR, result getting from quantitative PCR, non-polluting, high-throughput and easy to operate. The accuracy of detection clinical isolates consistent with mPCR, have good stability, which is appropriate for screening of a large number of clinical isolates of SP serotype.
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