| BackgroundStreptococcus 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.ObjectiveThis 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 results1. Established new multiplex fluorescence probes amplification for detection of Streptococcus pneumoniae serotypeFirst 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 serotypeIn 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 isolatesIn 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.ConclusionThe 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. |
PDF Full Text Request