Conventional polymerase chain reaction for the diagnosis of neurotoxoplasmosis: comparison of three sets of primers for the B1 gene using CSF samples

Polymerase chain reaction (PCR) has made a significant improvement in the diagnosis of toxoplasmic encephalitis (TE). Nevertheless, a wide variety of targets and primers has been used in different assays, and few comparative studies had been carried out. The aim of the present study was to compare the efficiency of 3 conventional PCR methods by using 3 sets of primers targeting the repetitive B1 gene in the diagnosis of TE. Diagnostic sensitivity and specificity of PCR and nested-PCR protocols were assessed for 207 (nested-PCR/T1-T4), 200 (nested-PCR/S1-AS1), and 206 (PCR/B22-B23) cerebrospinal fluid (CSF) samples, including AIDS and HIV-negative patients. The diagnostic sensitivity of PCR and nested-PCR assays was 50.85%, 68.97%, and 72.41% for T1-T4, S1-AS1, and B22-B23, respectively. The diagnostic specificity was high for all the assays showing values between 95% and 97%. In general, the best results were obtained for the B22-B23 set of primers, suggesting their usefulness compared with 2 nested-PCR protocols and showing that this simple and rapid strategy may be the preferred one for the diagnosis of TE in AIDS patients.     

Loop-mediated isothermal amplification assay targeting the omp25 gene for rapid detection of Brucella spp

A novel loop-mediated isothermal amplification (LAMP) assay was established to detect Brucella species DNA in milk and blood samples of animals and humans. This LAMP assay based on the sequence of highly repetitive omp25 gene was able to detect 9fg/μl Brucella spp. DNA with high sensitivity, which was 10 times higher than the nested PCR. The LAMP was evaluated for its specificity using 19 strains of six Brucella species and 28 related non-Brucella micro-organism strains as controls. The target 19 Brucella strains were all amplified, and no cross-reaction was found with all the non-Brucella micro-organism strains. Both nested PCR and LAMP assays were then used to detect Brucella spp. DNA in 78 milk samples and 113 blood samples from animals and 11 blood samples from humans, and the established LAMP assay yielded 99.0% concordance rate with the nested PCR. The LAMP assay should be a potential tool with high convenience, rapidity, sensitivity and specificity for the diagnosis of Brucellosis.     

黄疸出血群钩端螺旋体LAMP-LFB快速检测方法的建立与应用

  目的   基于环介导恒温扩增（LAMP）结合纳米生物传感条（LFB）技术,建立并评价黄疸出血群钩端螺旋体（钩体）的LAMP-LFB快速检测方法。  方法   以黄疸出血群钩体O抗原基因簇中的糖基转移酶基因（gtf）为检测靶标并设计特异性LAMP引物。 通过对LAMP引物的特定标记（FIP-FAM和LF-Biotin）和优化试验,建立LAMP-LFB快速检测方法,并分析其灵敏度和特异性。 应用建立的LAMP-LFB、普通PCR方法和显微凝集试验（MAT）,分别对53株钩体分离菌株进行鉴定,比较分析鉴定结果并评价LAMP-LFB方法的实用性。   结果   灵敏度和特异性试验显示,LAMP-LFB方法可检出黄疸出血群赖型56601基因组DNA的最低浓度为100 fg/μL,检测特异性为100%,与其余血清群和非钩体菌株核酸无交叉反应。 菌株鉴定结果显示,LAMP-LFB与MAT鉴定结果完全一致,符合率为100%,其敏感性高于PCR方法。 此外,LAMP扩增产物经LFB验证,可直接通过观察检测线（TL）和质控线（CL）进行结果判定。  结论   基于gtf基因建立的LAMP-LFB检测技术方便、快捷且重复性好,可快速、灵敏、特异地鉴定黄疸出血群钩体菌株,能够作为有价值的黄疸出血群钩体菌株快速鉴别或诊断方法。     

Detection of Toxoplasma gondii by polymerase chain reaction in cerebrospinal fluid from human immunodeficiency virus-1-infected Japanese patients with focal neurological signs

We aimed to determine the effectiveness of using the polymerase chain reaction (PCR) to detect Toxoplasma gondii in cerebrospinal fluid (CSF) specimens from Japanese patients infected with human immunodeficiency virus (HIV)-1. Twenty-six HIV-positive individuals presenting with focal neurological signs and a possible diagnosis of T. gondii encephalitis (TE) were enrolled in the study between April 1997 and March 2003. Eight patients were diagnosed as having TE using the accepted diagnostic criteria; PCR amplified the T. gondii B1 gene in CSF samples from five of these eight patients. CSF samples from the 18 patients without TE were negative for T. gondii DNA. The sensitivity, specificity and positive and negative predictive values for detecting T. gondii in CSF using PCR were 62.5%, 100%, 100% and 85.7%, respectively. These results suggest that PCR might be a clinically useful technique for detecting T. gondii DNA in patients infected with HIV showing focal neurological signs. Improvements in sensitivity are needed, however.     

LAMP技术检测非小细胞肺癌患者外周血EGFR基因L858R位点突变方法的建立及应用

目的利用环介导等温扩增(LAMP)技术建立非小细胞肺癌(NSCLC)患者外周血靶点表皮生长因子受体(EGFR)基因L858R位点突变的快速筛查方法。方法在Genebank上查找EGFR 21号外显子(L858R)的DNA序列,利用Primer Explorer V4软件设计特异性引物,建立LAMP检测方法,并对其特异性和敏感性进行评价。采集11例临床病理诊断为NSCLC患者外周血血浆,以聚合酶链反应(PCR)扩增结果为标准,对建立的LAMP方法的准确性进行验证。结果采用自行设计的LAMP引物及构建的反应体系可特异性扩增EGFR L858R位点突变阳性DNA,检测敏感性为0.1%,特异性达到100%。LAMP法在11例EGFR L858R位点突变阳性的NSCLC患者血浆标本中检测到9例阳性。结论成功建立了LAMP检测人外周血浆EGFR基因突变方法,诊断敏感性、特异性和准确性均较高,可荧光目测判读检测结果,是一种简单、快速的EGFR基因突变筛查方法。     

快速检测粪便中艰难梭菌的方法研究

目的建立可用于粪便中艰难梭菌快速检测的环介导恒温扩增(LAMP)方法。方法针对艰难梭菌毒素A基因,设计引物。采用LAMP对艰难梭菌和其他腹泻干扰菌及不同浓度艰难梭菌的扩增检测,对其特异性和灵敏度进行评价。同时采用LAMP和荧光定量PCR对100例疑似艰难梭菌感染的临床腹泻患者粪便进行检测,对两种方法进行比较。结果 LAMP对艰难梭菌及6种腹泻干扰菌的检测,只针对艰难梭菌有扩增,显示了较好的特异性。LAMP最低检测限为10CFU/mL,灵敏度较高。对100例临床粪便标本LAMP检测结果与荧光定量PCR比较差异无统计学意义(P>0.05,χ2=0.1429)。结论本研究建立的粪便中艰难梭菌的LAMP快速检测方法特异性好、灵敏度较高、操作简便,适用于艰难梭菌的临床快速检测及现场检测。     

Rapid diagnosis of septic arthritis using 16S rDNA PCR: a comparison of 3 methods

Few studies address the utility of molecular techniques for diagnosis of infection in synovial fluid (SF). We evaluated 3 different methods using 16S rDNA polymerase chain reaction (PCR) on 63 specimens for the diagnosis of joint infection. SF samples were classified as normal, inflammatory, or septic based on the patient's clinical and laboratory results. Samples were analyzed by conventional PCR using primers for the bacterial 16S rDNA gene and by real-time PCR utilizing 2 different sets of primers for the target gene 16S rDNA. PCR results were compared to culture results. All inflammatory and normal SF samples were culture negative. There was concordance with 10 of the 16 septic samples by 2 of the PCR methods. When comparing 3 methods for rapid detection of septic arthritis, real-time PCR using SYBR-Green I and conventional PCR demonstrated favorable test characteristics, but need further study.     

TaqMan荧光定量PCR检测新型隐球菌方法的建立

目的建立TaqMan荧光定量PCR方法定量检测新型隐球菌基因组DNA,为检测隐球菌性脑膜炎提供重要方法。方法在国家生物技术信息中心(NCBI)查找新型隐球菌各亚型的ITS-rDNA序列,序列比对后设计特异性引物和探针,扩增片段为114bp,构建质粒标准品,调整质粒浓度为1.42×10~8 copy/μL~1.42×10copy/μL共8个浓度梯度,分别取2μL作为模板,优化反应条件,建立标准曲线,进行敏感性、特异性、重复性评价,并检测临床确诊的15例隐球菌脑膜炎感染菌株。结果建立的荧光定量PCR可以检测2.84×10~2拷贝的质粒DNA,对临床分离的各10例其他真菌、细菌、乙型肝炎病毒DNA和人类基因组DNA均无扩增曲线,重复性良好,3个浓度的批间变异系数分别为2.86%、1.48%、1.36%,可准确检测15例新型隐球菌。结论成功建立检测新型隐球菌的荧光定量PCR方法,敏感性和特异性较高,结果稳定可靠,可早期、快速诊断隐球菌性脑膜炎。     

Polymerase chain reaction detection of Pneumocystis jiroveci: evaluation of 9 assays

Various polymerase chain reaction (PCR) amplification strategies have been described for detecting Pneumocystis jiroveci in clinical specimens. Different combinations of primer/target and platforms have been reported to yield varying PCR detection rates. PCR was evaluated on clinical specimens using internal transcribed spacer regions of the rRNA nested, dihydropteroate synthase single and nested, dihydrofolate reductase nested, major surface glycoprotein heminested, mitochondrial large subunit rRNA (mtLSUrRNA) single and nested, 18S rRNA 1-tube nested, and real-time 5S rRNA PCR. The most sensitive PCR was subsequently compared with routine diagnostic immunofluorescence (IF) microscopy. Discrepant PCR and IF results were resolved after review of clinical and histology/cytology records. Major discrepancies were observed among the methods investigated. mtLSUrRNA nested PCR was the most sensitive, produced less false-negative results, and displayed the highest degree of concordance with histology. Direct comparison of mtLSUrRNA nested PCR versus IF yielded low sensitivity and specificity, which were improved for PCR and lowered for IF on review of clinical and laboratory records.     

应用巢式PCR检测实验小鼠组织中申克孢子丝菌

目的研究实验小鼠申克孢子丝菌感染的分子生物学鉴定方法,为建立检测人申克孢子丝菌感染的快速、敏感、特异方法提供依据。方法建立申克孢子丝菌感染小鼠模型,应用合成的申克孢子丝菌种特异性引物ITS3-SSP进行巢式PCR扩增小鼠皮损组织内核糖体DNA的ITS2靶序列,将检测结果与标准形态学鉴定结果对比,观察种特异性引物PCR扩增结果的准确程度、敏感性和特异性。结果组织学检查可以从11只感染小鼠尾组织中见到染成紫红色的圆形、卵圆形孢子;第一循环PCR检测实验小鼠申克孢子丝菌,只有3只小鼠呈阳性;而巢式PCR检测申克孢子丝菌特异性DNA,11只实验小鼠中9只呈阳性;而3只对照组小鼠无一呈阳性。结论本实验结果说明巢式PCR结合种特异性引物ITS3-SSP可以有效检出实验小鼠皮损中的申克孢子丝菌,具有一定的敏感性和特异性,尤其是对于组织学检查和真菌培养阴性的标本,本方法是一个很有潜力的替代方法。     

乙型脑炎和登革热病毒的环介导等温扩增基因定量检测技术研究

目的建立一种适合口岸现场快速检测乙型脑炎和登革热病毒的环介导等温扩增(LAMP)定量技术。方法根据LAMP方法的原理,设计LAMP检测引物和反应体系,建立LAMP检测方法,同时综合评估初始拷贝数值与方法中的灵敏度、特异度、重复性及荧光信号值反应时间(1×104)之间的线性关联。结果检测选用1套LAMP引物,完成时间为0.5h,传统PCR检测与LAMP对比,差异明显,LAMP检测可有效提高灵敏度,是PCR检测技术的10倍。循环阈值和模版浓度具有良好的线性联系,实验室变异系数为小于5%。结论该方法是特异度与灵敏度较高,操作简单、结果判断容易、设备要求低的快速检测方法,适合基层医疗卫生机构和现场查验机构的广泛应用。     

环介导等温扩增技术快速检测副溶血性弧菌

目的建立一种检测副溶血性弧菌（Vp）快速且特异的环介导等温扩增（LAMP）检测方法。方法针对副溶血性弧菌不耐热溶血素（tlh）基因特异性序列的6个位点设计4条LAMP引物,65℃保温约60min,完成对副溶血性弧菌的扩增,扩增产物经肉眼、SYBR Green Ⅰ染色、电泳和酶切鉴定。利用LAMP和普通PCR方法同时检测1株副溶血性弧菌和12株非副溶血性弧菌来验证LAMP方法的特异性;将副溶血性弧菌菌液作一系列10倍稀释后用LAMP和PCR方法进行检测,比较两者敏感性。结果1株副溶血性弧菌出现LAMP扩增反应：通过肉眼、SYBR Green Ⅰ染色和电泳均能观察到LAMP扩增产物的出现,酶切证实了LAMP产物的特异性;12株非副溶血性弧菌未出现LAMP扩增。LAMP检测tlh基因的特异性和敏感性与普通PCR相同,LAMP检测tlh基因的检测下限为15cfu／mL。结论建立了一种快速、敏感、特异的副溶血性弧菌检测方法,适合日常监测及快速检测的需要。     

聚合酶链反应扩增STY3671基因鉴别伤寒与甲型副伤寒沙门菌

目的 评价伤寒沙门菌基因STY3671用于特异性检测该血清型沙门菌的可能性。 方法 根据伤寒沙门菌与甲型副伤寒沙门菌外膜蛋白质组比较得到的伤寒沙门菌特异性蛋白点编码基因STY3671的序列设计引物,提取伤寒沙门菌、甲型副伤寒沙门菌以及其他非伤寒沙门菌血清型菌株的基因组DNA进行PCR检测, 对引物特异性和灵敏性进行分析。 结果 检测的184株伤寒沙门菌均得到807 bp长度片段,部分菌株扩增产物测序结果与伤寒沙门菌标准株CT-18一致。146株甲型副伤寒沙门菌以及其余163株其他血清型沙门菌均未扩增出该片段。 结论 STY3671基因能够作为特异性检测伤寒沙门菌的靶标,能够区别于甲型副伤寒沙门菌及其他常见非伤寒沙门菌血清型,在发热患者的伤寒沙门菌感染诊断、尤其使用血液标本检测中具有良好的潜在价值。     

海洋创伤弧菌LAMP快速诊断方法的建立与评价

目的利用环介导等温扩增(LAMP)技术,建立海洋创伤弧菌快速、简便、特异且敏感的检测方法,并对该方法的特异性和灵敏度进行评价。方法选取海洋创伤弧菌溶细胞素(vvhA)基因作为靶基因,根据GenBank公布的序列设计4条LAMP引物;对47株细菌(包括20株弧菌属细菌)进行LAMP和聚合酶链式反应(PCR)扩增,并做特异性比较;对创伤弧菌M06株增菌液10倍倍比稀释,提取DNA后进行灵敏度的检测,并与常规PCR作比较;构建含vvhA基因片段的重组质粒,作为LAMP反应体系的标准阳性对照。结果常规PCR实验出现假阳性结果,而LAMP实验只有海洋创伤弧菌出现阳性扩增,其他样品均为阴性,无假阳性和假阴性结果,表明引物的特异性较好,加入钙黄绿素和电泳结果相一致;针对vvhA基因建立的LAMP技术其最低检测下限为每个反应4×10CFU,是常规PCR(每个反应4×10~2 CFU)的10倍,呈现较好的灵敏度。重复实验过程两遍,PCR和LAMP技术检测结果稳定。结论建立了一种用于检测海洋创伤弧菌的LAMP检测方法,该方法特异性强,灵敏度高,方便快捷,特别适合用于现场和床旁的快速检测。     

Application of loop-mediated isothermal amplification assay for the sensitive and rapid diagnosis of visceral leishmaniasis and post-kala-azar dermal leishmaniasis

Loop-mediated isothermal amplification (LAMP) is at the forefront in the search for innovative diagnostics for rapid and specific amplification of target DNA under isothermal conditions. We have applied LAMP assay using SYBR Green for clear-cut naked eye detection of Leishmania (Leishmania) donovani in 200 clinical samples of visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL). The assay was positive in 53/55 VL blood samples (sensitivity, 96.4%; 95% confidence interval [CI], 87.7–99%), 15/15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI, 79.6–100%), 60/62 PKDL tissue biopsy samples (sensitivity, 96.8%; 95% CI, 88.9–99.1%), and 1/68 control samples (specificity, 98.5%; 95% CI, 92.1–99.7%). The assay was specific for L. (L.) donovani, the causative species for VL and negative for L. (L.) infantum, L. (L.) tropica, and L. (L.) major. This is the first comprehensive clinical study demonstrating the applicability of the LAMP assay for a rapid and reliable molecular diagnosis of VL and PKDL.     

Rapid and sensitive detection of white spot syndrome virus by loop-mediated isothermal amplification combined with a lateral flow dipstick

Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acids under isothermal conditions using a set of four specifically designed primers that recognize six distinct target sequences. It can be combined with a chromatographic lateral flow dipstick (LFD) for highly specific, rapid and simple visual detection of WSSV-specific amplicons. Using this protocol, a 30-min amplification followed by 5 min hybridization with an FITC-labeled DNA probe and 5 min LFD resulted in visualization of DNA amplicons trapped at the LFD test line. Thus, 10 min for rapid DNA extraction followed by LAMP combined with LFD detection resulted in a total assay time of approximately 50 min. Detection sensitivity was comparable to other commonly-used methods for nested PCR detection of WSSV but had the additional advantages of reduced assay time, confirmation of amplicon identity by hybridization and elimination of electrophoresis with carcinogenic ethidium bromide.     

恙虫病东方体环介导等温扩增可视化快速检测方法的建立

目的 利用环介导等温扩增（LAMP）可视化检测技术,建立一种灵敏、特异和简便的恙虫病检测方法。方法 利用LAMP在线引物设计软件Primer Explorer V4,以恙虫病东方体热休克蛋白groEL基因作为靶序列,设计扩增引物,优化反应条件。利用恙虫病东方体标准株评估灵敏度和特异度;收集42份恙虫病血样,分别采用LAMP、普通PCR和实时荧光定量PCR方法进行验证。结果 建立的LAMP方法可在61～65℃80 min内完成恙虫病东方体的快速检测,最低检测限为10拷贝/μL,高于普通PCR方法 2个数量级,高于荧光定量PCR方法 1个数量级;对8种常见的自然疫源性疾病病原体检测均为阴性,特异度为100%;对42份恙虫病血样采用3种方法检测,阳性率一致,均为21.4%(9/42)。结论 本研究建立的恙虫病东方体LAMP可视化检测反应快速,操作简便,灵敏特异,可在基层医疗卫生机构运用推广。     

环介导等温扩增技术快速检测脑膜炎奈瑟菌属方法的建立

目的应用环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术建立一种快速敏感的脑膜炎奈瑟氏菌属检测方法。方法针对脑膜炎奈瑟菌属(ctrA)基因序列的6个区域设计4条LAMP引物(2条内引物、2条外引物),同时设计2条环引物,并对反应条件和反应体系进行优化。分别验证该方法的特异性及敏感性,并与普通PCR进行了比较。结果在适宜反应条件所设计引物对脑膜炎奈瑟菌的扩增的特异性及敏感性均较好,与普通PCR方法比较,LAMP敏感性比普通PCR高10倍。结论 LAMP检测速度相比PCR更快速,在60 min内即可完成扩增反应。实验建立的LAMP方法能够快速、灵敏、特异地检测脑膜炎奈瑟氏菌,适合基层检验部门及小型实验室与现场监测等使用。     

An improved primer design for the loop-mediated isothermal amplification (LAMP) method to detect oxacillinase (OXA)-48 β-lactamase genes in Gram-negative bacteria for clinical applications

IntroductionRecently, increased frequencies of carbapenemase-producing Enterobacteriaceae have been reported worldwide. Among multiple genetic subtypes, oxacillinase (OXA)-48 β-lactamase-producing strains have been associated with inbound infection because they have been detected predominantly in patients who traveled outside of Japan. However, a recent case report of OXA-48 β-lactamase-producing Enterobacteriaceae suggested the latent spread of domestic infections. Due to a lack of specific inhibitors, culture-based detection of OXA-48 β-lactamase-producing bacteria is difficult. Thus, DNA-based detection methods, including PCR, direct sequencing and loop-mediated isothermal amplification (LAMP), have been employed. Among these methods, LAMP detection is more favorable than other methods because of its technical simplicity and low cost.MethodsWe designed novel LAMP primers to detect OXA-48 β-lactamase-producing bacteria and investigated their possible clinical applications with bacterial genome-spiked human materials (cerebrospinal fluid, blood, feces, urine, and sputum). We evaluated the specificity of the LAMP primers using 37 bacterial strains: 8 standard, 9 reference, and 20 clinical Gram-negative strains.ResultsOur LAMP primers detected 10 copies of the OXA-48 type β-lactamase gene and exhibited no cross reactivity with other β-lactamase genes. Sensitivity was not influenced in any clinical sample, in contrast to PCR detection, which was strongly inhibited by substances in fecal samples.ConclusionsThese results suggest the superior performance of LAMP compared with conventional PCR for detecting the OXA-48 type β-lactamase gene in various clinical samples.     

武汉市流感病毒流行特征及检测方法的比较

目的 探讨武汉市甲、乙型流感病毒的流行特征,通过比较巢式PCR和胶体金法检测对流感病毒的检出情况,评价后者能否为临床流感样患者的流感初筛和诊断提供快速有效的方法.方法 采集2017—2019年武汉市流感样患者的鼻咽拭子,进行胶体金法检测,分析该地区甲、乙型流感病毒的流行病学特征;同时选取其中600份样本,进行巢式PCR...