Validation of the polymerase chain reaction for the diagnosis of human cutaneous leishmaniasis in north-west Colombia

Leishmania species of the subgenus Viannia account for 88% of all cases of leishmaniasis recorded in Colombia. Correct diagnosis is essential as infection with members of this subgenus can produce disfiguring destruction of the mucosa. Several methods are available to diagnose leishmaniasis in clinical samples. More recently, the polymerase chain reaction (PCR) has been used, with varying sensitivities and specificities depending on the primers used. In this paper we report on the sensitivity and specificity of PCR primers B1/B2 used on clinical samples and compare their use to the conventional parasitological methods. PCR alone is more sensitive than any single conventional method used, but a combination of conventional methods produced comparable sensitivity. PCR is well suited for use in selected cases and as a test for mucosal leishmaniasis.     

Detection of Leishmania kDNA in human serum samples for the diagnosis of visceral leishmaniasis

The performance of PCR to detect Leishmania kDNA in serum for the diagnosis of visceral leishmaniasis (VL) was assessed in serum samples from 65 patients with VL, 17 non-infected individuals and 17 patients with other febrile hepatosplenic diseases. Serum PCR showed a sensitivity of 85%, specificity of 100% and efficiency of 90%. The sensitivity values obtained for blood PCR (97%) and rK39 ELISA (95%) were significantly higher (P=0.01) than the values observed for L. chagasi ELISA (88%) and serum PCR (85%), whilst no difference was observed among the specificity rates obtained with rK39 ELISA (94%; P=0.47) and L. chagasi ELISA (85%; P=0.06). This work suggests that the use of serum samples may be an alternative for the diagnosis of VL when peripheral blood samples are not available or require significant operational efforts.     

逆转录-套式PCR检测汉坦病毒感染的实验研究

目的：探讨逆转录一套式聚合酶链反应（RT—nested—PCR）检测汉坦病毒（Hantavirus，HV）感染的准确性和敏感性。方法：用RT—nested—PCR和直接免疫荧光法（direct immunofluorescence，FA）分别检测深圳市2005年肾综合征出血热宿主动物监测中捕获的鼠类中HV的感染情况。结果：在76份特异性总抗体阳性的鼠肺标本中，用RT—nested—PCR检出61份抗原阳性，而FA仅检出47份阳性。结论：RT—nested—PCR是一种比较准确的检测汉坦病毒感染的方法，其敏感性高于FA。     

The detection of Plasmodium falciparum and P. vivax in DNA-extracted blood samples using polymerase chain reaction

Seventeen pairs of published primer sets were compared for their relative sensitivity to detect malaria DNA extracted from blood samples, which were obtained from Pakistani patients suffering from malaria. The primer sets investigated consisted of: (i) 9 pairs of direct primers and 3 sets of nested primers for detecting Plasmodium falciparum, (ii) 2 pairs of direct primers and 2 sets of nested primers for detecting P. vivax, and (iii) 1 set of multiplex primers for detecting both P. falciparum and P. vivax, simultaneously. After a miniscreen of 9 DNA-extracted blood samples using the 17 primer sets stated above, 5 primer sets were short-listed (based on their superior sensitivity) and used for a maxi-screen of DNA extracted from 126 microscopy-positive blood samples from Pakistan, with the following results. (i) For the detection of P. falciparum, the direct primer pair 'PF1 + PF2' gave a sensitivity of 95% and the nested primer set 'RIT405 + RIT406/RIT371 + RIT372' gave a sensitivity of 97%. (ii) For the detection of P. vivax, the direct primer pair 'Forward + Reverse' and the nested primer set 'PLF + UNR/PLF + VIR' both gave a sensitivity of 94%. (iii) The nested multiplex primer set 'rPLU5 + rPLU6/rFAL1 + rFAL2 + rVIV1 + rVIV2' gave a sensitivity of 97% and 96% for P. falciparum and P. vivax, respectively. It was concluded that the nested multiplex primer set was the most optimal primer set to use for the detection of malaria DNA extracted from blood samples. Furthermore, the nested multiplex primer set has the advantage of simultaneously detecting and differentiating between P. vivax and P. falciparum.     

Real-time polymerase chain reaction diagnosis of leishmaniasis in Panama from both fresh and frozen tissue

Skin biopsies stored in ethanol from 49 patients with suspected cutaneous leishmaniasis (CL) were tested in a real-time polymerase chain reaction (PCR) assay and compared with conventional diagnostic methods. With clinical diagnosis as the gold standard, PCR had a sensitivity of 96% (47/49) vs. 61% (30/49) for histopathology and 33% (16/49) for culture. In addition, DNA was extracted from 70 frozen smears of lesions from suspected cases of CL and tested with the same assay. In these samples, the PCR had a sensitivity of 61% (43/70) vs. 56% (39/70) for histopathology and 41% (29/70) for culture. In this study, real-time PCR offered a rapid diagnosis with an enhanced sensitivity over conventional methods. Although the yield of PCR diagnosis was lower when testing frozen smears, the assay still outperformed existing diagnostic modalities.     

A new PCR-ELISA for diagnosis of visceral leishmaniasis in blood of HIV-negative subjects

The PCR-ELISA represents a promising advance for diagnosis of visceral leishmaniasis (VL) in blood samples. However, the method has been validated mostly with HIV-positive patients who are known to have high levels of parasitaemia. We developed a new PCR-ELISA assay for specific detection of Leishmania in patients' blood and validated it in Nepalese subjects with clinically suspected VL, almost all of whom were HIV-negative. For blood samples, PCR-ELISA was more sensitive (83.9%) than conventional PCR (73.2%), and demonstrated 100% and 87.2% specificity when using healthy controls who had never travelled to a VL-endemic area and controls from a VL-endemic area as references, respectively. We have demonstrated the ability of PCR-ELISA to detect parasites in blood of HIV-negative patients. The method could be used for epidemiological as well as clinical purposes, as it reduces the need for traumatic bone marrow sampling and risky spleen aspiration.     

A nested polymerase chain reaction (Ln-PCR) for diagnosing and monitoring Leishmania infantum infection in patients co-infected with human immunodeficiency virus

We investigated a Leishmania-specific nested polymerase chain reaction (Ln-PCR) for the diagnosis and treatment monitoring of L. infantum infections in patients co-infected with human immunodeficiency virus (HIV). Peripheral blood and bone marrow samples from 89 HIV patients in Spain suspected of having leishmaniasis were examined by different diagnostic techniques (Ln-PCR, microscopy, NNN culture and indirect fluorescent antibody test). The sensitivity of Ln-PCR compared with microscopy and culture of bone marrow was 95.45% using blood and 100% when using bone marrow. 38 of these patients with confirmed leishmaniasis were entered in a chemotherapy trial (reported elsewhere), and samples from them were collected before treatment, one month after treatment ended and during follow-up (1-20 months), and examined similarly. Ln-PCR was shown to be a good method for testing efficacy of treatment and for predicting relapses after treatment (relapses were predicted on average 5 months earlier than when using classical diagnostic techniques). We suggest that Ln-PCR (especially using peripheral blood) should be the technique of choice for diagnosis, monitoring the success of treatment, and predicting relapses in patients with HIV and suspected or confirmed L. infantum infection.     

套式聚合酶链反应法检测外周血单个核细胞中HBcVccDNA

目的:建立套式聚合酶链反应法(nPCR)检测慢性乙型肝炎患者外周血单个核细胞(PBMC)中乙型肝炎病毒共价闭合环状DNA(HBV cccDNA)。方法:根据HBV基因组DNA正负链上下游缺口序列,设计两套相对保守的引物,建立HBV cccDNA套式PCR,并用该法检测200例慢性乙型肝炎患者PBMC中HBV cccDNA。结果:该法检测200例慢性乙型肝炎患者,HBV cccDNA阳性率为60%。结论:套式PCR法简便、灵敏,可用于测定PBMC中HBV cccDNA。     

套式聚合酶链反应法检测外周血单个核细胞中HBV cccDNA

目的：建立套式聚合酶链反应法（nPCR）检测慢性乙型肝炎患者外周血单个核细胞（PBMC）中乙型肝炎病毒共价闭合环状DNA（HBV cccDNA）。方法：根据HBV基因组DNA正负链上下游缺口序列，设计两套相对保守的引物，建立HBV cccDNA套式PCR，并用该法检测200例慢性乙型肝炎患者PBMC中HBV cccDNA。结果：该法检测200例慢性乙型肝炎患者，HBV cccDNA阳性率为60％。结论：套式PCR法简便、灵敏，可用于测定PBMC中HBV cccDNA。     

PCR检测胚外体腔液细胞DNA产前诊断的初步研究

目的对胚外体腔液DNA进行定量分析,初步判定胚外体腔液中细胞的数目;应用PCR方法对胚外体腔液细胞DNA进行检测,预测胎儿性别,初步建立一套孕早期检测伴性遗传疾病的方法.方法对25例孕6 -12周的孕妇在人流前行胚外体腔穿刺术,抽取胚外体腔液0.5-3ml,随后行人流术,取少量绒毛组织.采用煮沸法对25份胚外体腔液进行DNA抽提,Picogreen染料对25份胚外体腔液DNA进行定量.采用X,丫两对引物对25份胚外体腔液DNA进行PCR反应.绒毛组织进行染色体核型分析,与胚外体腔液的检测结果相对照.结果0.5 - 3 ml胚外体腔液含有750-6270个DNA拷贝(细胞).男性血DNA扩增结果出现两条带(X ,Y)女性血出现一条带(X).25份胚外体腔液样本11份出现两条带确定为男性胎儿,14份出现一条带确定为女性胎儿,与绒毛染色体核型分析结果一致.结论0.5 ml胚外体腔液DNA的含量就可满足PCR反应的要求.采用PCR技术对胚外体腔液细胞进行性别鉴定及伴性遗传病的研究,具有早期,快速,敏感的优点.     

Use of polymerase chain reaction in human African trypanosomiasis stage determination and follow-up.

Stage determination of human African trypanosomiasis is based on the detection of parasites and measurements of biological changes in the cerebrospinal fluid (CSF) (concentration of white blood cells > 5 cells per mm3 and increased total protein levels). The patient is treated accordingly. Demonstration of the absence or presence of trypanosomes by the double centrifugation technique is still the only test available to clinicians for assessing treatment success. In this study, however, we evaluate the polymerase chain reaction (PCR) as a tool for assessing the disease stage of trypanosomiasis and for determining whether treatment has been successful. All 15 study patients considered to be in the advanced stage of the disease were PCR positive; however, trypanosomes were demonstrated by double centrifugation in only 11 patients. Of the five remaining patients, who were considered to be in the early stage, PCR and double centrifugation were negative. Following treatment, 13 of the 15 second-stage patients were found to be negative for the disease in at least two samples by PCR and double centrifugation. Two others were still positive by PCR immediately and one month after the treatment. Trypanosome DNA detection using PCR suggested that the two positive patients were not cured but that their possible relapse could not be identified by a search for parasites using the double centrifugation technique. Further evaluation of the PCR method is required, in particular to determine whether PCR assays could be used in studies on patients who fail to respond to melarsoprol, as observed in several foci.     

套式聚合酶链反应技术检测孕妇外周血中胎儿细胞

目的探讨利用孕妇外周血中胎儿细胞进行产前诊断的可行性。方法采用套式聚合酶链反应(polymerase chain reaction，PCR)技术扩增孕妇外周血中男性胎儿性别决定基因(sexdetermining region Y，SRY)，并与胎儿实际性别相对照。结果74例孕7～40周的孕妇中38例怀男胎，其中23例扩增出Y特异带；另外36例怀女胎孕妇中，仅1例出现Y特异带。SRY套式PCR扩增系统在孕中期，孕晚期检查孕妇外周血中男胎灵敏度(72．7％、71．4％)、特异度(100．0％)和符合率(87．0％、90．5％)高，误诊率为0，而在孕早期灵敏度(50．0％)、特异度(90．0％)及符合率(63．3％)较低，误诊率(10．0％)、漏诊率(50．0％)较高。结论胎儿细胞在孕早期即存在于孕妇外周血中，但直接利用孕妇外周血中胎儿细咆检查SRY基因的检出率较低。     

用PCR技术测定按蚊人血指数的研究

目的建立一种能替代传统免疫学方法用于按蚊人血指数测定的分子生物学检测技术。方法根据人核糖体DNA序列设计1对特异性引物,建立聚合酶链反应（PCR）鉴定按蚊胃内人血的方法。同时,对猪血、牛血、羊血、小鼠血以及未吸血按蚊中提取的DNA进行检测,验证该检测方法的特异性,并对吸饲人血后不同时间（1、6、12、18、24、27、30、33、36、40、4J4、48h）的按蚊进行检测,测试该方法的检测敏感性。结果该方法可从人血提取的DNA中扩增得到519bp大小的特异性条带,对其他动物血样及未吸血按蚊中所提取的DNA均未能扩增出特异性条带;所有吸人血24h内的中华按蚊均能扩增出特异性条带,在吸人血后27、30、33、36h的各5只中华按蚊中,分别有4、4、2、1只能扩出特异性条带,吸血40h后的中华按蚊均不能扩增出特异性条带。Logistic回归分析表明,吸血后24-40h,PCR检测阳性按蚊数与吸血后的时间呈负相关关系（P〈0．01）。结论本研究所建立的PCR方法可准确鉴定吸血24h内的中华按蚊胃内的人血液来源,可替代传统免疫学方法用于按蚊人血指数的测定。     

实时定量PCR检测人全血标本中烟曲霉基因组载量的方法及临床应用

目的 建立实时定量PCR(RQ-PCR)快速检测人全血标本中烟曲霉基因组载量的方法及进行初步临床应用.方法 基于烟曲霉多拷贝基因ITS1-5.8S基因设计引物和TaqMan探针,用QIAamp(R)DNA Blood Mini Kit提取烟曲霉基因组DNA,建立20μl RQ-PCR反应体系,对含有不同载量烟曲霉基因组的模拟人全血标本和66份外科发热患者全血标本进行烟曲霉基因组的定量检测.结果 检测限为10-1基因组/μl上机待测液(即约78 CFU/ml全血);检测特异度和灵敏度分别为94.25%和99.04%,阳性预告值和阴件预告值分别为97.63%和97.62%;测定结果的平均相对误差为(3.67±13.19)%;批内及批间平均重复性变异系数分别为(12.38±1.53)%和(16.27±2.72)%;人血标本中烟曲霉基因组平均回收率为(107.81±25.92)%,回收率平均变异系数为(26.24±5.62)%.66份外科发热患者血标本中未检测出烟曲霉基因组.结论 RQ-PCR可以快速、特异、灵敏地定量检测人血标本中烟曲霉基因组的载量,且有着较好的准确度与精密度.本研究外科发热患者血中未检测到烟曲霉基因组.

Abstract：

Objective To establish a real-time quantitative polymerase chain reaction (RQ-PCR) assay for fast detection of Aspergillus fumigatus genome in human whole blood samples and explore its clinical application.Methods The primers and the TaqMan-probe were designed on the basis of the multi-copy ITS1-5. 8S region of the rDNA of Aspergillus fumigatus. The Aspergillus fumigatus genomic DNA were extracted with QIAamp(R) DNA Blood Mini Kit.A 20 μl RQ-PCR amplification system was established, and the simulated blood samples containing various given load of Aspergillus fumigatus genome and the 66 whole blood samples of the surgical febrile patients were examined. Results The detection limit of the RQ-PCR instrument is 10-1 genomes/μl DNA sample,namely 78 CFU/ml whole blood. The specificity and the sensitivity were 94. 25% and 99. 04% respectively; and the positive predictive value and negative predictive value were 97. 63% and 97. 62% respectively. The average relative error of the quantitative results was (3. 67 ±13. 19)%, and the intra- and the inter-assay average coefficients of variation were (12.38 ± 1. 53)% and (16. 27 ±2. 72)% , respectively. The average recovery rate of Aspergillus fumigatus genomic DNA in human whole blood samples was (107. 81 ±25. 92)% , and the average coefficient of variation of the average recovery rate was (26. 24 ± 5.62) % . No Aspergillus fumigatus genomic DNA was detected among the 66 blood samples of the surgical febrile patients. Conclusions The RQ-PCR assay for fast quantitative detection of Aspergillus fumigatus genome in human whole blood samples is of high sensitivity, specificity,accuracy and precision. The Aspergillus fumigatus genome was not detected in this group of surgical febrile patients.     

优化的套式核酸体外扩增方法用于甲型流感病毒快速诊断的研究

目的临床上对流感的有效控制与治疗在于实验室快速与准确的诊断,这对于指导用药和避免滥用抗生素具有重要意义。RT_PCR在流感病毒检测中特异性和灵敏度较强,nestedmultiplexRT_PCR法可以在此基础上提高灵敏度。实验的目的是应用分子生物学方法对流感病毒进行快速诊断。方法我们结合流感病毒M基因、NS基因、HA基因的结构和进化特性设计多元引物达到流感病毒的型和亚型准确与快速的鉴定。结果甲型流感病毒以M基因保守区域设计引物,PCR后电泳带为413bp;甲型流感病毒以HA基因分亚型,H1N1亚型PCR后电泳带为348bp,H3N2亚型PCR后电泳带为291bp,H5N1亚型PCR后电泳带为326bp。结论应用优化的套式核酸体外扩增方法进行流感病毒的型与亚型的诊断是比较快速的,且所需要的病毒量少(0.01～0.1TCID50),灵敏度较高。     

巢式聚合酶链反应对霍乱弧菌肠毒素快速检测方法的建立

[目的]　建立霍乱弧菌肠毒素快速检测的检验技术。[方法]　运用巢式聚合酶链反应(NESTED-PCR)检测霍乱弧菌肠毒素基因CTX。[结果]　检测霍乱弧菌特异性强,检测下限达4cfu/mL(100cfu/mL级水平)。[结论]　NESTED-PCR检定霍乱弧菌简便、快速、敏感度高且准确性好。     

MassTag polymerase chain reaction for differential diagnosis of viral hemorrhagic fever

Viral hemorrhagic fevers are associated with high rates of illness and death. Although therapeutic options are limited, early differential diagnosis has implications for containment and may aid in clinical management. We describe a diagnostic system for rapid, multiplex polymerase chain reaction identification of 10 different causes of viral hemorrhagic fevers.     

巢式甲基化特异性PCR检测p16基因甲基化

目的利用巢式甲基化特异性PCR（Nested-MSP,nMSP）法检测非小细胞肺癌患者血清中p16基因的甲基化状态,探讨其在肿瘤早期诊断中的意义。方法利用nMSP法检测了65例非小细胞肺癌患者（其中鳞癌48例,腺癌17例）血清中p16基因的甲基化状态。结果在65例非小细胞肺癌患者血清中,p16基因的甲基化百分率为72．3％（47／65）;nMSP法比普通的MSP法具有更高的灵敏度。结论巢式甲基化特异性PCR是一种灵敏度高、特异性强的甲基化检测方法,可广泛应用于基因甲基化分析。