人类粪便中小肠结肠炎耶尔森菌检测方法的研究
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摘要
研究背景
感染性腹泻(infectious diarrhea)由各种病原微生物及其产物或者寄生虫所引起,不仅对人的身体健康危害较大,也严重影响了公共卫生,带来一系列社会和经济问题。及时对感染性腹泻致病病原体进行快速、灵敏、特异性地检测和诊断,是科学预防和控制感染性腹泻暴发和流行的重要保证。
小肠结肠炎耶尔森菌(Yersinia enterocolitica, Y. enterocolitica)是近二十年来引起国际上广泛关注的一种重要的感染性腹泻致病菌,大多数人类患者通过消化道受到感染。人类感染该菌后,主要表现为发热、腹痛、腹泻等急性胃肠炎的症状,还可引起结节性红斑、活动性关节炎、耶氏肝炎,其他还有脑膜炎、心肌炎、虹膜炎、深部脓肿、败血症及创伤感染等肠道外症状,甚至还能由超抗原引起突眼性甲状腺肿等自身免疫性疾病。目前普遍用于小肠结肠炎耶尔森菌检测的方法有细菌分离培养和生化鉴定、普通PCR法、Real-time PCR法等。传统的分离培养和生化鉴定还是诊断的“金标准”,然而其敏感性不高,而且耗时耗力。以PCR法为基础的分子生物学技术则存在实验设备昂贵,检测费用较高、对检测人员技术要求较高等缺点,使其不适用于现场快速检测和基层单位推广普及使用。如何建立新的快速、简便的检测方法成为研究的热点。
环介导等温扩增(loop-mediated isothermal amplification, LAMP)技术是近年来新出现和新发展起来的一种敏感、特异、方便快捷的链置换核酸扩增技术,可作为病原体所致的感染性疾病早期检测和鉴定的一种简单而快速的诊断工具。整个过程非常简单而且迅速,仅需在一个反应管内加入所有反应试剂,利用针对目标基因设计的特异性LAMP引物,在恒温的条件下即可完成。该技术已经用于多种细菌、病毒、真菌和寄生虫的检测,其优势在于等温扩增,整个反应过程简单,而扩增效率更高,还能保证反应的高灵敏度和特异度,通过肉眼观察或者琼脂糖凝胶电泳即可判定反应结果,尤其适合于病原体的现场快速检测、战时野外环境下及基层单位普及应用。
最近,有学者应用LAMP技术检测食品中的小肠结肠炎耶尔森菌,获得了较好的灵敏度和特异度,但是该技术能否用于粪便中小肠结肠炎耶尔森菌的检测以及检测效果如何,目前国内外尚无报道。
此外,现有的研究显示,绝大多数学者在进行LAMP条件优化试验时,习惯性地按照全面试验法、多次单因素法依次优化反应条件,繁琐费时,还不一定能够得到最佳的扩增效果,对影响LAMP反应的主要因素也未见定量分析的报道。
系统评价(Systematic review)是循证医学的重要研究方法。它针对某一个具体的临床或科研问题进行定性或定量分析,获得指导临床或者科研的最佳证据。90年代以来,国内外已发表数以千计的有关系统评价的论文,涉及医学研究的各个领域,包括病因研究、诊断性试验、防治评价、预后研究等等。迄今为止,有关普通PCR法、Real-time PCR法等核酸扩增技术检测小肠结肠炎耶尔森菌的文献报道较多,但未见系统评价用于这些检测方法的综合分析。
研究目的
1.对目前已有报道的主要核酸扩增技术(普通PCR法、Real-time PCR法和LAMP法)检测小肠结肠炎耶尔森菌的研究进行系统评价以了解这些技术当前的检测情况；
2.建立粪便标本中的小肠结肠炎耶尔森菌的LAMP检测方法,并将正交设计引入LAMP反应的条件优化试验中,进一步对影响LAMP反应的主要因素进行分析；
3.对优化的LAMP法的特异度和灵敏度等指标进行测试,并将其用于临床粪便样本的实际检测,同时作分离培养、普通PCR和荧光定量PCR平行检测,比较不同检测方法,对所建立LAMP法进行应用评价。
研究方法
1.制定原始文献的纳入、排除标准及检索策略,检索MEDLINE、EMBASE、BIOSIS、Web of Science、Cochrane Library以及万方学术期刊全文数据库、维普中文期刊数据库(华师站)、中国知网(日期1990.01.01.-2010.12.31)。纳入的文献采用QUADAS(Quality assessment of Diagnostic Accuracy Studies)进行质量评价,用MetaDisc 1.4软件进行数据分析。
2.针对致病性小肠结肠炎耶尔森菌特有的粘附侵袭位点基因(ail基因),利用在线软件Primer Explorer version 4设计LAMP引物。根据正交表L16(45)共进行16组处理,每组重复2次,使用直观对比法、极差分析和方差分析来确定最佳扩增反应体系,并对影响LAMP反应扩增效果的主要因素进行分析；
3.利用NCBI的Blast分析比对LAMP所使用内、外引物的特异度,并对3株小肠结肠炎耶尔森菌菌株和18株非耶尔森菌其他细菌菌株进行LAMP扩增检测所建立LAMP反应的特异度。从细菌基因组DNA、纯培养菌落、模拟感染粪便标本三个方面对LAMP法的灵敏度进行检测,同时作普通PCR和荧光定量PCR平行检测与之比较。此外,同时使用分离培养法、普通PCR法、荧光定量PCR法和LAMP法对206例腹泻患者粪便标本和50例正常粪便标本中的致病性小肠结肠炎耶尔森菌进行检测,比较了四种方法的检测情况。
结果
1.符合标准的共有11篇文献,12个研究。采用随机效应模型进行数据分析。合并后的诊断优势比(DOR)为458.91(95%CI：72.62-2900.09),敏感度为0.72(95%CI：0.69-0.75),特异度为0.96(95%CI：0.95-0.96),阳性似然比为27.23(95%CI：10.60-69.96),阴性似然比为0.09(95%CI：0.02-0.30)。合并后的准确性测量指标均具有高度异质性(P<0.1,12>50%),合并没有意义,但不存在阈值效应所致异质性。
2.针对致病性小肠结肠炎耶尔森菌特有的粘附侵袭位点基因(ail基因)成功设计了4条LAMP引物。根据正交表L16(45)完成16次试验并重复2次后,经过直观对比、极差分析和方差分析最终确立最佳反应体系,即在25μl反应体系中,内引物FIP、BIP各0.8μM,外引物F3、B3各0.2μM, 1.0M Betaine, 1.0mM dNTPs,4.0mM MgC12,8U Bst DNA聚合酶,2.5μl 10×Thermopol缓冲液和5μlDNA模板。扩增程序为：60℃孵育60 min,然后80℃孵育4min。经实践证明,该LAMP反应体系重复性好、可靠稳定。方差分析结果显示,本试验条件下反应体系中五个主要因素对LAMP扩增效果影响从大到小依次为：FIP/BIP、dNTPs、Mg2+、F3/B3、Betaine。
3.(1)NCBI的Blast比对结果发现,研究中所设计的内引物FIP/BIP和外引物B3/F3均与小肠结肠炎耶尔森菌具有100%的同源性。采用LAMP方法对21株细菌的基因组DNA扩增,除所有的小肠结肠炎耶尔森菌检测阳性外,其它对照细菌检测结果均为阴性,表明该检测方法特异度好。
(2)以小肠结肠炎耶尔森菌标准菌株为试验菌株,灵敏度试验显示,该LAMP法最低可检测低达约为5.3fg(电泳检测或紫外透射仪下肉眼检测)或53fg/25μl反应体系(自然光下肉眼检测)小肠结肠炎耶尔森菌基因组DNA,对于细菌纯培养物检测极限约为1.5 CFU/ml(电泳检测或紫外透射仪下肉眼检测)或15 CFU/ml(自然光下肉眼检测),直接对粪便样本中的小肠结肠炎耶尔森菌进行检测的极限为15CFU/g(电泳检测或紫外透射仪下肉眼检测)或150 CFU/g(自然光下肉眼检测)。与同时进行的普通PCR和Real-time PCR相比,普通PCR法与自然光下肉眼检测LAMP结果的灵敏度一致,比电泳检测或紫外透射仪下肉眼检测结果的灵敏度低,Real-time PCR的最低检测度则与电泳检测或紫外透射仪下肉眼检测结果一致,培养法需要菌液浓度达到1.5×103CFU/g才能从粪便标本中分离到细菌。即LAMP法比传统PCR的灵敏度高了10倍,与Real-time PCR一致,相对细菌培养计数法,LAMP法的灵敏度则要高出2个数量级。
(3)206例腹泻患者粪便标本LAMP检测后,成功检出11例样本中含有致病性小肠结肠炎耶尔森菌,与普通PCR和Real-time PCR完全符合,但经培养和鉴定成功的为9例。50例正常对照者中,四种检测方法均未检出致病性小肠结肠炎耶尔森菌。以培养法为“金标准”,LAMP法的特异性为99.0%,培养阳性的9例样本经LAMP法检测也均为阳性,Youden指数为0.990。经McNemar检验,两种检测方法之间的差异无显著性(P=0.500),二者吻合度较强。(Kappa系数=0.895,P=0.000)
结论
1.总的来说,普通PCR法、Real-time PCR法和LAMP法检测小肠结肠炎耶尔森菌的诊断效能较高,可是各个研究的异质性大,测量指标敏感度(9%-100%)、特异度(37%-100%)等的变化范围也较大,尚不能取代传统的培养方法单独用于检测小肠结肠炎耶尔森菌,但可以联合传统的培养方法进行检测,尚需进行大规模的流行病学调查以评价这些核酸扩增方法。
2.本研究针对致病性小肠结肠炎耶尔森菌ail基因成功建立LAMP检测方法。经优化的LAMP法特异度强,灵敏度高,使用恒温水浴锅1小时即可完成,不需要昂贵精密的仪器和特殊的试剂,可通过多种方法实现对终点产物的检测,不同检测方法造成检测的灵敏度有所差异。
3.该LAMP法适用于粪便样本中小肠结肠炎耶尔森菌的检测。粪便标本检测的过程包括了过夜冷增菌、DNA提取以及LAMP扩增和结果判定,全部步骤只需不到1天即可完成。用于实际样本检测时,与培养法、普通PCR法和Real-time PCR法显示了良好的一致性。
4.正交试验与方差分析相结合优化LAMP反应体系,比单因素试验、全面试验的方法简单快捷,比简单的直观对比、极差分析更准确可靠,适用于LAMP反应体系的快速优化。
5. FIP/BIP、F3/B3、dNTPs、Mg2+、Betaine浓度对LAMP扩增效果均有影响,各因素影响效果大小依次为：FIP/BIP、dNTPs、Mg2+、F3/B3、Betaine。
Backgrounds
Infectious diarrhea which is caused by a wide variety of pathogenic microorganisms and their metabolites or parasites does great harm to human health, and also has a severe impact on public health, bringing a series of social and economic problems. Rapid, sensitive and specific detection and diagnosis for pathogens that lead to diarrheal disease in time are of great significance to the prevention and control of outbreaks and prevalence of infectious diarrhea.
Yersinia enterocolitica, an important pathogen of infectious diarrhea, has been of great concern worldwide in recent years and intake of contaminated food may be the leading transmission route contributing to human infection. Infection with Yersinia enterocolitica in humans causes various diseases, and the most common one is acute gastroenteritis with the main clinical manifestations of fever, abdominal pain and diarrhea. Besides, it may be the causal agent of extraintestinal symptoms including erythema nodosum, reactive arthritis, granulomatous hepatitis, meningitis, myocarditis, iritis, deep abscess, septicemia, wound infection and sometimes even contribute to autoimmune disorder like Graves' disease via superantigen. Routine detection of suspected individuals for Yersinia enterocolitica infection is based upon bacteria isolation, and subsequent biochemical test identification, as well as PCR assay and Real-time PCR assay. The conventional isolation method has been considered as the gold standard for diagnosis, but the whole identification process is laborious, time consuming and less sensitive. PCR-based molecular biological methods (PCR or real-time PCR) require expensive instruments, high detection cost and skilled personnel, which may delay their application in field rapid detection and in peripheral health care settings and clinics. A rapid and simple new detection method for Yersinia enterocolitica is significantly required, and has become the hot spot of research.
Loop mediated isothermal amplification (LAMP) is a sensitive, specific and convenient strand- displacement gene amplification technique newly emerging as a simple and rapid diagnostic tool for early detection and identification of microbial diseases in recent years.
The whole procedure is very simple and rapid wherein the amplification employs a set of specifically designed primers of a target gene and can be completed by incubating all the reagents in a single tube under isothermal conditions. The LAMP method has successfully detected many kinds of pathogenic organisms, including bacteria, viruses, fungi and parasites. Considering its advantages of isothermal amplification, simple operation, high amplification efficiency, easy detection by agarose gel electrophoresis or visual observation together with high sensitivity and specificity, LAMP has potential applications for pathogen detection especially in field rapid detection, open country detection during wartime and peripheral health care settings and clinics.
Recent studies on the detection of Yersinia enterocolitica by LAMP have focused merely on contaminated foods, though great sensitivity and specificity were achieved. Whether the method can be applied in feces and what the detection results are have not been reported in China or in other countries until now.
The LAMP reaction was traditionally optimized by the comprehensive-test method or one-factor-at-a-time method by a majority of researchers. These methods usually involve a relatively large number of experiments which are labour intensive, and time-consuming, but they may not be able to completely guarantee the determination of the optimal conditions. In addition, there have not been any reports on quantifying analysis of the main factors influencing the LAMP reaction.
Systematic review is an important method of Evidence-Based Medicine, in which qualitational and quantificational analyses are conducted on a specific clinical or research issue in the purpose of acquiring the best evidence for guidance. Thousands of papers about systematic review involving all areas of medical research have been published, including etiopathogenisis, diagnostic tests, prevention and treatment evaluation, prognosis and so on. So far, there have been many reports on Yersinia enterocolitica detection by nucleic acid amplification including PCR, Real-time PCR. Nevertheless, no systematic review has been found to evaluate these methods.
Objectives
The present study aimed to:
1. Synthetically appraise the studies using systematic review on Yersinia enterocolitica detection by nucleic acid amplification(PCR, Real-time PCR and LAMP) which have already been reported at present;
2. develop a LAMP method for detecting Yersinia enterocolitica in human feces, optimize LAMP reaction condition based on orthogonal design and then further analyze the main factors that would influence LAMP reaction;
3. determine the specificity and sensitivity of the optimized LAMP method, which was also applied in clinical faecal samples. The conventional isolation method, PCR assay and Real-time PCR assay were conducted parallelly and compared with the developed LAMP method to evaluate its application.
Methods
1. MEDLINE, EMBASE, BIOSIS, Web of Science, Cochrane Library, WANFANG DATA, VIP INFORMATION, CNKI (Jan.1st,1990-Dec.31st,2010) were searched on studies of the main nucleic acid amplification technology for the detection of Yersinia enterocolitica. Quality of included literatures was assessed by QUADAS (quality assessment of diagnostic accuracy studies). The software Meta Disc1.4 was used to summarize the data.
2. LAMP primers directing ail gene specific for pathogenic Yersinia enterocolitica were designed using the online software Primer Explorer version 4. According to the orthogonal table L16(45),16 sets of treatment repeated twice, a total of 32 experiments were investigated, and the optimal amplification reaction system was determined in the employment of direct comparison, range analysis and variance analysis. The main factors that affect the LAMP amplification were analyzed simultaneously;
3. Blast of NCBI was utilized to test the specificity of LAMP primers including the outer primers and inner primers.3 strains of Yersinia enterocolitica, and 18 non-Yersinia other bacterial strains were also examined to further assure specificity of the established LAMP method. Sensitivity of the LAMP method was evaluated in Yersinia enterocolitica genome DNA, pure culture bacteria and artificially infected faecal specimens, with PCR and Real-time PCR as parallel detection at the same time. In addition,206 faecal specimens from diarrheal patients and 50 normal specimens were detected for pathogenic Yersinia enterocolitica by culture, PCR, Real-time PCR and LAMP in order to make a comparison among them.
Results
1.12 relevant studies from 11 literatures were selected according to the standards. Data synthesis was performed using random effect model. Pooled accuracy indicators of DOR(diagnostic odds ratio), sensitivity, specificity, positive LR (likelihood ratio) and negative LR were 458.91 (95%CI:72.62-2900.09),0.72 (95%CI:0.69-0.75),0.96 (95%CI:0.95-0.96),27.23(95%CI:10.60-69.96) and 0.09(95%CI:0.02-0.30) respectively. High heterogeneity was found and all these summary measures of diagnostic accuracy were not clinically meaningful(P<0.1, I2>50%), but there was no threshold effect.
2. Four LAMP primers were designed corresponding to the ail gene specific for pathogenic Yersinia enterocolitica. The optimal LAMP condition was containing 0.8μM each inner primer(FIP and BIP),0.2μM each outer primer(F3 and B3), 1.0mM dNTPs,4.0mM MgCl2,1.0M Betaine,8U of Bst DNA polymerase, 2.5μl of 10×Thermopol buffer and 5μl DNA template in 25μl volumes, which was confirmed by direct comparison, range analysis and variance analysis after 32 LAMP reactions were completed according to the orthogonal table L16(45). And the amplification program was as follows:60℃for 60 min and then heated at 80℃for 4min. The optimized LAMP reaction system was proved to be repeatable, reliable and stable. Results from analysis of variance displayed that the order of amplification effect of the five main factors were FIP/BIP, dNTPs, Mg2+, F3/B3, Betaine in turn under conditions of the present study.
3. (1)Both the designed inner primer FIP/BIP and outer primer B3/F3 were found to have 100% homology with Yersinia enterocolitica by Blast of NCBI. Only genomic DNA from Yersinia enterocolitica strains was positively detected by LAMP, while no LAMP products were obtained when detecting non-Yersinia strains, which showed the good specificity of the method.
(2)When the reference strain was used as the test organism, the LAMP method can detect Yersinia enterocolitica genomic DNA as low as 5.3fg (observed by gel electrophoresis or visual detection under UV light) or 53fg (visual detection under normal light)/25μl reaction system minimally; When detecting template DNA from pure culture bacteria, the LAMP method was capable of detecting 1.5 CFU/ml (observed by gel electrophoresis or visual detection under UV light) or 15 CFU/ml (visual detection under normal light) at the least; in artificially infected faecal specimens, the detection limit of the LAMP method was 15 CFU/g (observed by gel electrophoresis or visual detection under UV light) or 150 CFU/g (visual detection under normal light). In comparison, it was possible for PCR to detect the target sequence with the same detection limit as LAMP investigated by visual inspection under normal light. However, PCR was found to be less sensitive than LAMP observed by gel electrophoresis or visual detection under UV light. Real-time PCR had the same detection limit as LAMP observed by gel electrophoresis or visual detection under UV light. The culture method can isolate the bacteria at concentrations above 1.5×103CFU/g from feces. Thus, LAMP here we introduced was 10 times more sensitive than PCR, had the same detection limit as Real-time PCR, and was 100 times more sensitive compared to culture method.
4. (3) With regard to the 206 clinical specimens from diarrhea patients,11 were tested positive using the LAMP assay by visual detection or agarose gel electrophoresis, which were in accordance with PCR and Real-time PCR. But only 9 of them were isolated and identified successfully by the culture method. All the four methods failed to detect pathogenic Yersinia enterocolitica in 50 normal feces from healthy individuals. The specificity of LAMP was calculated to be 99.0% with culture as the "gold standard", all of the nine culture-positive specimens were also positive for LAMP and the Youden index was 0.990. The two methods showed no significant difference (P=0.500) and had great agreement with each other (Kappa=0.895, P=0.000)
Conclusions
1. Generally, PCR, Real-time PCR and the LAMP assay for detection of Yersinia enterocolitica had a high test performance, but there was great heterogeneity among each study. Also, these accuracy measures like sensitivity (9%-100%) and specificity (37%-100%) had an extremely wide range. Therefore, these methods can not be used alone for Yersinia enterocolitica detection in place of traditional culture method, but the application of them combined with the culture method was recommended. And large epidemiological surveys were still required to assess these methods.
2. A LAMP method was successfully developed to detect the pathogenic Yersinia enterocolitica directing ail gene in the present study. The optimized LAMP method was proved to be sensitive and specific, and can be completed in one hour using thermostatic water bath without elaborate and expensive instruments and special reagents. Also, the amplification results can be observed by several endpoint detection methods, but there was some difference in the detection limits.
3. The established LAMP method was suitable for Yersinia enterocolitica detection in faecal samples. The detection process in feces comprised the cold enrichment overnight, DNA extraction together with LAMP amplification and result identification, which took less than 1 day to be accomplished. The new method showed a satisfactory concordance with the conventional culture method, PCR and real-time PCR method when being applied in clinical specimens.
4. Orthogonal design combined with variance analysis was easy to operate and time-saving in the optimization of LAMP reaction system compared with comprehensive-test method or single-factor method and was more accurate and reliable than the simple analytical methods of direct comparison and range analysis. So the novel method was a considerable method to optimize LAMP reaction system.
5. FIP/BIP, F3/B3, dNTPs, Mg2+ and Betaine concentrations all played important roles in LAMP amplification, and the order of effects of those factors was FIP/ BIP, dNTPs, Mg2+, F3/B3 and Betaine.
感染性腹泻(infectious diarrhea)由各种病原微生物及其产物或者寄生虫所引起,不仅对人的身体健康危害较大,也严重影响了公共卫生,带来一系列社会和经济问题。及时对感染性腹泻致病病原体进行快速、灵敏、特异性地检测和诊断,是科学预防和控制感染性腹泻暴发和流行的重要保证。
小肠结肠炎耶尔森菌(Yersinia enterocolitica, Y. enterocolitica)是近二十年来引起国际上广泛关注的一种重要的感染性腹泻致病菌,大多数人类患者通过消化道受到感染。人类感染该菌后,主要表现为发热、腹痛、腹泻等急性胃肠炎的症状,还可引起结节性红斑、活动性关节炎、耶氏肝炎,其他还有脑膜炎、心肌炎、虹膜炎、深部脓肿、败血症及创伤感染等肠道外症状,甚至还能由超抗原引起突眼性甲状腺肿等自身免疫性疾病。目前普遍用于小肠结肠炎耶尔森菌检测的方法有细菌分离培养和生化鉴定、普通PCR法、Real-time PCR法等。传统的分离培养和生化鉴定还是诊断的“金标准”,然而其敏感性不高,而且耗时耗力。以PCR法为基础的分子生物学技术则存在实验设备昂贵,检测费用较高、对检测人员技术要求较高等缺点,使其不适用于现场快速检测和基层单位推广普及使用。如何建立新的快速、简便的检测方法成为研究的热点。
环介导等温扩增(loop-mediated isothermal amplification, LAMP)技术是近年来新出现和新发展起来的一种敏感、特异、方便快捷的链置换核酸扩增技术,可作为病原体所致的感染性疾病早期检测和鉴定的一种简单而快速的诊断工具。整个过程非常简单而且迅速,仅需在一个反应管内加入所有反应试剂,利用针对目标基因设计的特异性LAMP引物,在恒温的条件下即可完成。该技术已经用于多种细菌、病毒、真菌和寄生虫的检测,其优势在于等温扩增,整个反应过程简单,而扩增效率更高,还能保证反应的高灵敏度和特异度,通过肉眼观察或者琼脂糖凝胶电泳即可判定反应结果,尤其适合于病原体的现场快速检测、战时野外环境下及基层单位普及应用。
最近,有学者应用LAMP技术检测食品中的小肠结肠炎耶尔森菌,获得了较好的灵敏度和特异度,但是该技术能否用于粪便中小肠结肠炎耶尔森菌的检测以及检测效果如何,目前国内外尚无报道。
此外,现有的研究显示,绝大多数学者在进行LAMP条件优化试验时,习惯性地按照全面试验法、多次单因素法依次优化反应条件,繁琐费时,还不一定能够得到最佳的扩增效果,对影响LAMP反应的主要因素也未见定量分析的报道。
系统评价(Systematic review)是循证医学的重要研究方法。它针对某一个具体的临床或科研问题进行定性或定量分析,获得指导临床或者科研的最佳证据。90年代以来,国内外已发表数以千计的有关系统评价的论文,涉及医学研究的各个领域,包括病因研究、诊断性试验、防治评价、预后研究等等。迄今为止,有关普通PCR法、Real-time PCR法等核酸扩增技术检测小肠结肠炎耶尔森菌的文献报道较多,但未见系统评价用于这些检测方法的综合分析。
研究目的
1.对目前已有报道的主要核酸扩增技术(普通PCR法、Real-time PCR法和LAMP法)检测小肠结肠炎耶尔森菌的研究进行系统评价以了解这些技术当前的检测情况；
2.建立粪便标本中的小肠结肠炎耶尔森菌的LAMP检测方法,并将正交设计引入LAMP反应的条件优化试验中,进一步对影响LAMP反应的主要因素进行分析；
3.对优化的LAMP法的特异度和灵敏度等指标进行测试,并将其用于临床粪便样本的实际检测,同时作分离培养、普通PCR和荧光定量PCR平行检测,比较不同检测方法,对所建立LAMP法进行应用评价。
研究方法
1.制定原始文献的纳入、排除标准及检索策略,检索MEDLINE、EMBASE、BIOSIS、Web of Science、Cochrane Library以及万方学术期刊全文数据库、维普中文期刊数据库(华师站)、中国知网(日期1990.01.01.-2010.12.31)。纳入的文献采用QUADAS(Quality assessment of Diagnostic Accuracy Studies)进行质量评价,用MetaDisc 1.4软件进行数据分析。
2.针对致病性小肠结肠炎耶尔森菌特有的粘附侵袭位点基因(ail基因),利用在线软件Primer Explorer version 4设计LAMP引物。根据正交表L16(45)共进行16组处理,每组重复2次,使用直观对比法、极差分析和方差分析来确定最佳扩增反应体系,并对影响LAMP反应扩增效果的主要因素进行分析；
3.利用NCBI的Blast分析比对LAMP所使用内、外引物的特异度,并对3株小肠结肠炎耶尔森菌菌株和18株非耶尔森菌其他细菌菌株进行LAMP扩增检测所建立LAMP反应的特异度。从细菌基因组DNA、纯培养菌落、模拟感染粪便标本三个方面对LAMP法的灵敏度进行检测,同时作普通PCR和荧光定量PCR平行检测与之比较。此外,同时使用分离培养法、普通PCR法、荧光定量PCR法和LAMP法对206例腹泻患者粪便标本和50例正常粪便标本中的致病性小肠结肠炎耶尔森菌进行检测,比较了四种方法的检测情况。
结果
1.符合标准的共有11篇文献,12个研究。采用随机效应模型进行数据分析。合并后的诊断优势比(DOR)为458.91(95%CI：72.62-2900.09),敏感度为0.72(95%CI：0.69-0.75),特异度为0.96(95%CI：0.95-0.96),阳性似然比为27.23(95%CI：10.60-69.96),阴性似然比为0.09(95%CI：0.02-0.30)。合并后的准确性测量指标均具有高度异质性(P<0.1,12>50%),合并没有意义,但不存在阈值效应所致异质性。
2.针对致病性小肠结肠炎耶尔森菌特有的粘附侵袭位点基因(ail基因)成功设计了4条LAMP引物。根据正交表L16(45)完成16次试验并重复2次后,经过直观对比、极差分析和方差分析最终确立最佳反应体系,即在25μl反应体系中,内引物FIP、BIP各0.8μM,外引物F3、B3各0.2μM, 1.0M Betaine, 1.0mM dNTPs,4.0mM MgC12,8U Bst DNA聚合酶,2.5μl 10×Thermopol缓冲液和5μlDNA模板。扩增程序为：60℃孵育60 min,然后80℃孵育4min。经实践证明,该LAMP反应体系重复性好、可靠稳定。方差分析结果显示,本试验条件下反应体系中五个主要因素对LAMP扩增效果影响从大到小依次为：FIP/BIP、dNTPs、Mg2+、F3/B3、Betaine。
3.(1)NCBI的Blast比对结果发现,研究中所设计的内引物FIP/BIP和外引物B3/F3均与小肠结肠炎耶尔森菌具有100%的同源性。采用LAMP方法对21株细菌的基因组DNA扩增,除所有的小肠结肠炎耶尔森菌检测阳性外,其它对照细菌检测结果均为阴性,表明该检测方法特异度好。
(2)以小肠结肠炎耶尔森菌标准菌株为试验菌株,灵敏度试验显示,该LAMP法最低可检测低达约为5.3fg(电泳检测或紫外透射仪下肉眼检测)或53fg/25μl反应体系(自然光下肉眼检测)小肠结肠炎耶尔森菌基因组DNA,对于细菌纯培养物检测极限约为1.5 CFU/ml(电泳检测或紫外透射仪下肉眼检测)或15 CFU/ml(自然光下肉眼检测),直接对粪便样本中的小肠结肠炎耶尔森菌进行检测的极限为15CFU/g(电泳检测或紫外透射仪下肉眼检测)或150 CFU/g(自然光下肉眼检测)。与同时进行的普通PCR和Real-time PCR相比,普通PCR法与自然光下肉眼检测LAMP结果的灵敏度一致,比电泳检测或紫外透射仪下肉眼检测结果的灵敏度低,Real-time PCR的最低检测度则与电泳检测或紫外透射仪下肉眼检测结果一致,培养法需要菌液浓度达到1.5×103CFU/g才能从粪便标本中分离到细菌。即LAMP法比传统PCR的灵敏度高了10倍,与Real-time PCR一致,相对细菌培养计数法,LAMP法的灵敏度则要高出2个数量级。
(3)206例腹泻患者粪便标本LAMP检测后,成功检出11例样本中含有致病性小肠结肠炎耶尔森菌,与普通PCR和Real-time PCR完全符合,但经培养和鉴定成功的为9例。50例正常对照者中,四种检测方法均未检出致病性小肠结肠炎耶尔森菌。以培养法为“金标准”,LAMP法的特异性为99.0%,培养阳性的9例样本经LAMP法检测也均为阳性,Youden指数为0.990。经McNemar检验,两种检测方法之间的差异无显著性(P=0.500),二者吻合度较强。(Kappa系数=0.895,P=0.000)
结论
1.总的来说,普通PCR法、Real-time PCR法和LAMP法检测小肠结肠炎耶尔森菌的诊断效能较高,可是各个研究的异质性大,测量指标敏感度(9%-100%)、特异度(37%-100%)等的变化范围也较大,尚不能取代传统的培养方法单独用于检测小肠结肠炎耶尔森菌,但可以联合传统的培养方法进行检测,尚需进行大规模的流行病学调查以评价这些核酸扩增方法。
2.本研究针对致病性小肠结肠炎耶尔森菌ail基因成功建立LAMP检测方法。经优化的LAMP法特异度强,灵敏度高,使用恒温水浴锅1小时即可完成,不需要昂贵精密的仪器和特殊的试剂,可通过多种方法实现对终点产物的检测,不同检测方法造成检测的灵敏度有所差异。
3.该LAMP法适用于粪便样本中小肠结肠炎耶尔森菌的检测。粪便标本检测的过程包括了过夜冷增菌、DNA提取以及LAMP扩增和结果判定,全部步骤只需不到1天即可完成。用于实际样本检测时,与培养法、普通PCR法和Real-time PCR法显示了良好的一致性。
4.正交试验与方差分析相结合优化LAMP反应体系,比单因素试验、全面试验的方法简单快捷,比简单的直观对比、极差分析更准确可靠,适用于LAMP反应体系的快速优化。
5. FIP/BIP、F3/B3、dNTPs、Mg2+、Betaine浓度对LAMP扩增效果均有影响,各因素影响效果大小依次为：FIP/BIP、dNTPs、Mg2+、F3/B3、Betaine。
Backgrounds
Infectious diarrhea which is caused by a wide variety of pathogenic microorganisms and their metabolites or parasites does great harm to human health, and also has a severe impact on public health, bringing a series of social and economic problems. Rapid, sensitive and specific detection and diagnosis for pathogens that lead to diarrheal disease in time are of great significance to the prevention and control of outbreaks and prevalence of infectious diarrhea.
Yersinia enterocolitica, an important pathogen of infectious diarrhea, has been of great concern worldwide in recent years and intake of contaminated food may be the leading transmission route contributing to human infection. Infection with Yersinia enterocolitica in humans causes various diseases, and the most common one is acute gastroenteritis with the main clinical manifestations of fever, abdominal pain and diarrhea. Besides, it may be the causal agent of extraintestinal symptoms including erythema nodosum, reactive arthritis, granulomatous hepatitis, meningitis, myocarditis, iritis, deep abscess, septicemia, wound infection and sometimes even contribute to autoimmune disorder like Graves' disease via superantigen. Routine detection of suspected individuals for Yersinia enterocolitica infection is based upon bacteria isolation, and subsequent biochemical test identification, as well as PCR assay and Real-time PCR assay. The conventional isolation method has been considered as the gold standard for diagnosis, but the whole identification process is laborious, time consuming and less sensitive. PCR-based molecular biological methods (PCR or real-time PCR) require expensive instruments, high detection cost and skilled personnel, which may delay their application in field rapid detection and in peripheral health care settings and clinics. A rapid and simple new detection method for Yersinia enterocolitica is significantly required, and has become the hot spot of research.
Loop mediated isothermal amplification (LAMP) is a sensitive, specific and convenient strand- displacement gene amplification technique newly emerging as a simple and rapid diagnostic tool for early detection and identification of microbial diseases in recent years.
The whole procedure is very simple and rapid wherein the amplification employs a set of specifically designed primers of a target gene and can be completed by incubating all the reagents in a single tube under isothermal conditions. The LAMP method has successfully detected many kinds of pathogenic organisms, including bacteria, viruses, fungi and parasites. Considering its advantages of isothermal amplification, simple operation, high amplification efficiency, easy detection by agarose gel electrophoresis or visual observation together with high sensitivity and specificity, LAMP has potential applications for pathogen detection especially in field rapid detection, open country detection during wartime and peripheral health care settings and clinics.
Recent studies on the detection of Yersinia enterocolitica by LAMP have focused merely on contaminated foods, though great sensitivity and specificity were achieved. Whether the method can be applied in feces and what the detection results are have not been reported in China or in other countries until now.
The LAMP reaction was traditionally optimized by the comprehensive-test method or one-factor-at-a-time method by a majority of researchers. These methods usually involve a relatively large number of experiments which are labour intensive, and time-consuming, but they may not be able to completely guarantee the determination of the optimal conditions. In addition, there have not been any reports on quantifying analysis of the main factors influencing the LAMP reaction.
Systematic review is an important method of Evidence-Based Medicine, in which qualitational and quantificational analyses are conducted on a specific clinical or research issue in the purpose of acquiring the best evidence for guidance. Thousands of papers about systematic review involving all areas of medical research have been published, including etiopathogenisis, diagnostic tests, prevention and treatment evaluation, prognosis and so on. So far, there have been many reports on Yersinia enterocolitica detection by nucleic acid amplification including PCR, Real-time PCR. Nevertheless, no systematic review has been found to evaluate these methods.
Objectives
The present study aimed to:
1. Synthetically appraise the studies using systematic review on Yersinia enterocolitica detection by nucleic acid amplification(PCR, Real-time PCR and LAMP) which have already been reported at present;
2. develop a LAMP method for detecting Yersinia enterocolitica in human feces, optimize LAMP reaction condition based on orthogonal design and then further analyze the main factors that would influence LAMP reaction;
3. determine the specificity and sensitivity of the optimized LAMP method, which was also applied in clinical faecal samples. The conventional isolation method, PCR assay and Real-time PCR assay were conducted parallelly and compared with the developed LAMP method to evaluate its application.
Methods
1. MEDLINE, EMBASE, BIOSIS, Web of Science, Cochrane Library, WANFANG DATA, VIP INFORMATION, CNKI (Jan.1st,1990-Dec.31st,2010) were searched on studies of the main nucleic acid amplification technology for the detection of Yersinia enterocolitica. Quality of included literatures was assessed by QUADAS (quality assessment of diagnostic accuracy studies). The software Meta Disc1.4 was used to summarize the data.
2. LAMP primers directing ail gene specific for pathogenic Yersinia enterocolitica were designed using the online software Primer Explorer version 4. According to the orthogonal table L16(45),16 sets of treatment repeated twice, a total of 32 experiments were investigated, and the optimal amplification reaction system was determined in the employment of direct comparison, range analysis and variance analysis. The main factors that affect the LAMP amplification were analyzed simultaneously;
3. Blast of NCBI was utilized to test the specificity of LAMP primers including the outer primers and inner primers.3 strains of Yersinia enterocolitica, and 18 non-Yersinia other bacterial strains were also examined to further assure specificity of the established LAMP method. Sensitivity of the LAMP method was evaluated in Yersinia enterocolitica genome DNA, pure culture bacteria and artificially infected faecal specimens, with PCR and Real-time PCR as parallel detection at the same time. In addition,206 faecal specimens from diarrheal patients and 50 normal specimens were detected for pathogenic Yersinia enterocolitica by culture, PCR, Real-time PCR and LAMP in order to make a comparison among them.
Results
1.12 relevant studies from 11 literatures were selected according to the standards. Data synthesis was performed using random effect model. Pooled accuracy indicators of DOR(diagnostic odds ratio), sensitivity, specificity, positive LR (likelihood ratio) and negative LR were 458.91 (95%CI:72.62-2900.09),0.72 (95%CI:0.69-0.75),0.96 (95%CI:0.95-0.96),27.23(95%CI:10.60-69.96) and 0.09(95%CI:0.02-0.30) respectively. High heterogeneity was found and all these summary measures of diagnostic accuracy were not clinically meaningful(P<0.1, I2>50%), but there was no threshold effect.
2. Four LAMP primers were designed corresponding to the ail gene specific for pathogenic Yersinia enterocolitica. The optimal LAMP condition was containing 0.8μM each inner primer(FIP and BIP),0.2μM each outer primer(F3 and B3), 1.0mM dNTPs,4.0mM MgCl2,1.0M Betaine,8U of Bst DNA polymerase, 2.5μl of 10×Thermopol buffer and 5μl DNA template in 25μl volumes, which was confirmed by direct comparison, range analysis and variance analysis after 32 LAMP reactions were completed according to the orthogonal table L16(45). And the amplification program was as follows:60℃for 60 min and then heated at 80℃for 4min. The optimized LAMP reaction system was proved to be repeatable, reliable and stable. Results from analysis of variance displayed that the order of amplification effect of the five main factors were FIP/BIP, dNTPs, Mg2+, F3/B3, Betaine in turn under conditions of the present study.
3. (1)Both the designed inner primer FIP/BIP and outer primer B3/F3 were found to have 100% homology with Yersinia enterocolitica by Blast of NCBI. Only genomic DNA from Yersinia enterocolitica strains was positively detected by LAMP, while no LAMP products were obtained when detecting non-Yersinia strains, which showed the good specificity of the method.
(2)When the reference strain was used as the test organism, the LAMP method can detect Yersinia enterocolitica genomic DNA as low as 5.3fg (observed by gel electrophoresis or visual detection under UV light) or 53fg (visual detection under normal light)/25μl reaction system minimally; When detecting template DNA from pure culture bacteria, the LAMP method was capable of detecting 1.5 CFU/ml (observed by gel electrophoresis or visual detection under UV light) or 15 CFU/ml (visual detection under normal light) at the least; in artificially infected faecal specimens, the detection limit of the LAMP method was 15 CFU/g (observed by gel electrophoresis or visual detection under UV light) or 150 CFU/g (visual detection under normal light). In comparison, it was possible for PCR to detect the target sequence with the same detection limit as LAMP investigated by visual inspection under normal light. However, PCR was found to be less sensitive than LAMP observed by gel electrophoresis or visual detection under UV light. Real-time PCR had the same detection limit as LAMP observed by gel electrophoresis or visual detection under UV light. The culture method can isolate the bacteria at concentrations above 1.5×103CFU/g from feces. Thus, LAMP here we introduced was 10 times more sensitive than PCR, had the same detection limit as Real-time PCR, and was 100 times more sensitive compared to culture method.
4. (3) With regard to the 206 clinical specimens from diarrhea patients,11 were tested positive using the LAMP assay by visual detection or agarose gel electrophoresis, which were in accordance with PCR and Real-time PCR. But only 9 of them were isolated and identified successfully by the culture method. All the four methods failed to detect pathogenic Yersinia enterocolitica in 50 normal feces from healthy individuals. The specificity of LAMP was calculated to be 99.0% with culture as the "gold standard", all of the nine culture-positive specimens were also positive for LAMP and the Youden index was 0.990. The two methods showed no significant difference (P=0.500) and had great agreement with each other (Kappa=0.895, P=0.000)
Conclusions
1. Generally, PCR, Real-time PCR and the LAMP assay for detection of Yersinia enterocolitica had a high test performance, but there was great heterogeneity among each study. Also, these accuracy measures like sensitivity (9%-100%) and specificity (37%-100%) had an extremely wide range. Therefore, these methods can not be used alone for Yersinia enterocolitica detection in place of traditional culture method, but the application of them combined with the culture method was recommended. And large epidemiological surveys were still required to assess these methods.
2. A LAMP method was successfully developed to detect the pathogenic Yersinia enterocolitica directing ail gene in the present study. The optimized LAMP method was proved to be sensitive and specific, and can be completed in one hour using thermostatic water bath without elaborate and expensive instruments and special reagents. Also, the amplification results can be observed by several endpoint detection methods, but there was some difference in the detection limits.
3. The established LAMP method was suitable for Yersinia enterocolitica detection in faecal samples. The detection process in feces comprised the cold enrichment overnight, DNA extraction together with LAMP amplification and result identification, which took less than 1 day to be accomplished. The new method showed a satisfactory concordance with the conventional culture method, PCR and real-time PCR method when being applied in clinical specimens.
4. Orthogonal design combined with variance analysis was easy to operate and time-saving in the optimization of LAMP reaction system compared with comprehensive-test method or single-factor method and was more accurate and reliable than the simple analytical methods of direct comparison and range analysis. So the novel method was a considerable method to optimize LAMP reaction system.
5. FIP/BIP, F3/B3, dNTPs, Mg2+ and Betaine concentrations all played important roles in LAMP amplification, and the order of effects of those factors was FIP/ BIP, dNTPs, Mg2+, F3/B3 and Betaine.
引文
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