Sensitive detection of Shiga Toxin 2 and some of its variants in environmental samples by a novel immuno-PCR assay

Shiga toxin-producing Escherichia coli (STEC) in the environment has been reported frequently. However, robust detection of STEC in environmental samples remains difficult because the numbers of bacteria in samples are often below the detection threshold of the method. We developed a novel and sensitive immuno-PCR (IPCR) assay for the detection of Shiga toxin 2 (Stx2) and Stx2 variants. The assay involves immunocapture of Stx2 at the B subunit and real-time PCR amplification of a DNA marker linked to a detection antibody recognizing the Stx2 A subunit. The qualitative detection limit of the assay is 0.1 pg/ml in phosphate-buffered saline (PBS), with a quantification range of 10 to 100,000 pg/ml. The IPCR method was 10,000-fold more sensitive than an analogue conventional enzyme-linked immunosorbent assay (ELISA) in PBS. Although the sensitivity of the IPCR for detection of Stx2 was affected by environmental sample matrices of feces, feral swine colons, soil, and water from watersheds, application of the IPCR assay to 23 enriched cultures of fecal, feral swine colon, soil, and watershed samples collected from the environment revealed that the IPCR detected Stx2 in all 15 samples that were shown to be STEC positive by real-time PCR and culture methods, demonstrating a 100% sensitivity and specificity. The modification of the sandwich IPCR we have described in this study will be a sensitive and specific screening method for evaluating the occurrence of STEC in the environment.     

Detection of rViscumin in plasma samples by immuno-PCR

To allow for pharmacokinetic studies in adjunction with the current clinical developments of the potent cytostatic anti-cancer drug rViscumin, a sandwich immuno-PCR (IPCR) assay was developed for the detection of rViscumin in blood plasma. The IPCR was carried out with a commercially available reagent kit, consisting of pre-assembled rViscumin-specific antibody-DNA conjugates as well as a specific competitor DNA fragment to be amplified by PCR. Various combinations of capture- and detection-antibodies were compared for performance in IPCR. Using the optimized assay, as few as 50 zeptomol (approx. 100 fg/ml) rViscumin (MW 57 kDa) was detectable in standardized human serum samples. The IPCR assay was very selective for rViscumin and in spiking experiments in proband plasma samples, signal recovery rates between 70% and 120% were obtained. The linear sensitivity range of the assay covered more than five orders of magnitude. Repeated measurements of rViscumin resulted in a mean standard deviation value of 14.2%.     

Applications of real-time immuno-polymerase chain reaction (rt-IPCR) for the rapid diagnoses of viral antigens and pathologic proteins

One of the major challenges of performing the IPCR has been to establish a robust, sensitive, and specific method which is easily adapted and highly standardized for routine use in a clinical laboratory. Presently, the performance of IPCR typically involves elaborate and multiple, time-consuming steps prone to high variation in reagents and technical application. Further advances in the technology and instrumentation used for the signal detection of IPCR has resulted in the development of real-time IPCR (rt-IPCR). Rt-IPCR is still relatively undeveloped in comparison to the use of both real-time PCR and IPCR as evidenced by the low number (eight citations) of publications in the scientific literature. However, increased use of rt-IPCR has shown that the method displays improved statistical validation of accuracy over IPCR. Inter-assay error is typically 5-10% vs 15-20% for IPCR. The primary advantage of using rt-IPCR in place of IPCR is the immediate interpretation of positive data (quantification of proteins) as the PCR reaction proceeds. This aspect is key to real-time diagnosis and has great importance for specific emergency situations (i.e., biological and environmental contaminations of toxins in biothreat situations), as well as cases where specific tumor/viral antigens and pathologic proteins may be present in body tissues in extremely low concentrations and rapid, early diagnosis is important for immediate palliative treatment. This review summarizes all of the experimental data published to date utilizing the rt-IPCR method for various analytes (vascular endothelial growth factor, mumps Ag, rViscumin, various IgG, gliadin, HIV-1 p24 Ag, Rotavirus VP6, pathologic and recombinant prion, and prostate specific Ag) and describes the molecular scaffold formats, solid formats, instrument detection systems, and probes/primers or fluorescent dyes used in these assays. With further standardization and validation, rt-IPCR has the potential to become the most analytically sensitive method available for the detection of proteins.     

大熊猫轮状病毒PCR检测方法的建立及应用

轮状病毒是威胁大熊猫健康的主要病原微生物之一。为了对大熊猫轮状病毒进行快速、方便且准确地检测,研发适合于基层饲养单位和保护区的检测方法是非常有必要的。本研究通过合成大熊猫轮状病毒Vp7基因序列,构建了PUC-VP7的重组质粒,并将其作为阳性对照对大熊猫轮状病毒样本进行PCR检测和分析。结果表明,在进行PCR扩增分析时,该质粒和病毒cDNA二者均在340 bp处出现了特异性条带。此外,对收集到的45份大熊猫粪便样本进行轮状病毒抗原检测时,其中2份样品在340 bp处出现条带,该基因片段与大熊猫轮状病毒CH-1株的相似性为99.89%。本研究构建的PUC-VP7质粒不但可以作为大熊猫轮状病毒PCR检测中的阳性质控品,而且还能有效地促进该PCR病毒检测技术在基层饲养单位和保护区的推广和应用。     

Detection of ricin and other ribosome-inactivating proteins by an immuno-polymerase chain reaction assay

Ribosome-inactivating proteins (RIPs) are plant proteins with enzymatic activity, classified as type 1 (single chain) or type 2 (two chains). They are identified as rRNA N-glycosidases (EC 3.2.2.22) and cause an irreversible inhibition of protein synthesis. Among type 2 RIPs, there are potent toxins (ricin is the best known) that are considered as potential biological weapons. The development of a fast and sensitive method for the detection of biological agents is an important tool to prevent or deal with the consequences of intoxication. In this article, we describe a very sensitive immuno-polymerase chain reaction (IPCR) assay for the detection of RIPs-a type 1 RIP (dianthin) and a type 2 RIP (ricin)-that combines the specificity of immunological analysis with the exponential amplification of PCR. The limit of detection (LOD) of the technique was compared with the LODs of the conventional immunological methods enzyme-linked immunosorbent assay (ELISA) and fluorescent immunosorbent assay (FIA). The LOD of IPCR was more than 1 million times lower than that of ELISA, allowing the detection of 10 fg/ml of dianthin and ricin. The possibility to detect ricin in human serum was also investigated, and a similar sensitivity was observed (10 fg/ml). IPCR appears to be the most sensitive method for the detection of ricin and other RIPs.     

A highly sensitive immuno-PCR assay for detection of H5N1 avian influenza virus

With an aim at detecting the ultra-low concentration of avian influenza virus (AIV), a highly sensitive hybrid assay based on immunology and polymerase chain reaction was developed. The TopYield microtiter plates were coated with ten-fold serial dilutions of H5N1 subtype AIV ranging from 10 EID₅₀ ml⁻¹~10⁻⁴ EID₅₀ ml⁻¹,which was recognized by mouse anti-AIV H5 monoclonal antibody (MAb) that was directly linked with reporter DNA using a heterobifunctional cross-linker. After extensive washing, the reporter DNA including a BamH I-restriction site was released by a specific enzymatic restriction, then transferred to PCR tubes, amplified, and used as the signal for detection of AIV. Under the optimized condition, MAb-based immuno-PCR (IPCR) method could measure 100 μl of AIV H5N1 with 10⁻⁴ EID₅₀ ml⁻¹.To evaluate the sensitivity of IPCR, the same concentration and volume of AIV H5N1 were detected by conventional RT–PCR and sandwich ELISA. The results showed that IPCR had an approximately 1,000-fold improvement over the conventional ELISA, and a 100-fold enhancement compared with RT–PCR in detection sensitivity. To further evaluate the specificity of IPCR for AIV H5 subtype, the tracheal swab specimens, taken from chickens which were infected with H9N2, and the allantoic fluid from eggs inoculated by AIV H3N2, H7N1, H9N2, were detected by IPCR. To mimic clinical samples, pharyngeal–tracheal swab specimens were collected from healthy chickens and spiked with H5N1, H5N2, H5N3 for analysis by immuno-PCR. The results demonstrated that IPCR was a highly sensitive and specific assay for AIV H5, and could be applied to clinical detection for low amount of AIV H5 subtype. This MAb-based immuno-PCR method provided a platform capable of mass screening of clinical samples for AIV H5 subtype and could serve as a model for other immuno-PCR assays.     

Immuno capture PCR for rapid and sensitive identification of pathogenic Bacillus anthracis

Immuno capture PCR (IPCR) is a technique capable of detecting the pathogens with high specificity and sensitivity. Rapid and accurate detection of Bacillus anthracis was achieved using anti-EA1 antibodies to capture the cells and two primer sets targeting the virulence factors of the pathogen i.e., protective antigen (pag) and capsule (cap) in an IPCR format. Monoclonal antibodies specific to B. anthracis were generated against extractable antigen 1 protein and used as capture antibody onto 96 well polystyrene plates. Following the binding of the pathogen, the DNA extraction was carried out in the well itself and further processed for PCR assay. We compared IPCR described here with conventional duplex PCR using the same primers and sandwich ELISA using the monoclonal antibodies developed in the present study. IPCR was capable of detecting as few as 10 and 100 cfu ml^?1 of bacterial cells and spores, respectively. IPCR was found to be 2–3 logs more sensitive than conventional duplex PCR and the sandwich ELISA. The effect of other bacteria and any organic materials on IPCR was also analyzed and found that this method was robust with little change in the sensitivity in the presence of interfering agents. Moreover, we could demonstrate a simple process of microwave treatment for spore disruption which otherwise are resistant to chemical treatments. Also, the IPCR could clearly distinguish the pathogenic and nonpathogenic strains of B. anthracis in the same assay. This can help in saving resources on unnecessary decontamination procedures during false alarms.     

Retrieval of entire genes from environmental DNA by inverse PCR with pre-amplification of target genes using primers containing locked nucleic acids

We had been unsuccessful to amplify desired nucleotide sequences from various environmental DNA samples by using the inverse polymerase chain reaction (IPCR) technique, most probably because the copy numbers of target DNA sequences had been quite low. To enrich the target DNA sequences prior to IPCR, a rolling-circle amplification was used with a site-specific primer containing locked nucleic acids (LNAs). This pre-amplified IPCR (PAI-PCR) method increased the sensitivity of PCR almost 10 000 times compared with the standard IPCR in model experiments using Escherichia coli . We then applied the PAI-PCR method to isolate glycosyl hydrolase genes from DNAs extracted from vermiform appendixes of horses and termite guts. The flanking sequences of the target genes were amplified and cloned successfully using PAI-PCR, whereas standard IPCR resulted in no amplification.     

Detecting antigens by quantitative immuno-PCR

The quantitative immuno-PCR (qIPCR) technology combines the advantages of flexible and robust immunoassays with the exponential signal amplification power of PCR. The qIPCR allows one to detect antigens using specific antibodies labeled with double-stranded DNA. The label is used for signal generation by quantitative PCR. Because of the efficiency of nucleic acid amplification, qIPCR typically leads to a 10- to 1,000-fold increase in sensitivity compared to an analogous enzyme-amplified immunoassay. A standard protocol of a qIPCR assay to detect human interleukin 6 (IL-6) using a sandwich immunoassay combined with real-time PCR readout is described here. The protocol includes initial immobilization of the antigen, and coupling of this antigen with antibody-DNA conjugates is then carried out by (a) the stepwise assembly of biotinylated antibody, streptavidin and biotinylated DNA, (b) the use of a biotinylated antibody and an anti-biotin-DNA conjugate or (c) the employment of an anti-IL-6 antibody-DNA conjugate. Following the assembly of signal-generating immunocomplexes, real-time PCR is used to amplify and record the signal. Depending on the coupling strategy, the qIPCR assays require 4-7 h with only about 3 h hands-on-time. The use of qIPCR assays enables the detection of rare biomarkers in complex biological samples that are poorly accessible by conventional immunoassays. Therefore, qIPCR offers novel opportunities for the biomedical analysis of, for instance, neurodegenerative diseases and viral infections as well as new tools for the development of novel pharmaceuticals.     

Simultaneous detection of waterborne viruses by multiplex real-time PCR

Norovirus, Rotavirus group A, the Hepatitis A virus, and Coxsackievirus are all common causes of gastroenteritis. Conventional diagnoses of these causative agents are based on antigen detection and electron microscopy. To improve the diagnostic potential for viral gastroenteritis, internally controlled multiplex real-time polymerase chain reaction (PCR) methods have been recently developed. In this study, individual real-time PCRs were developed and optimized for specific detections of Norovirus genogroup I, Norovirus genogroup II, Rotavirus group A, the Hepatitis A virus, and Coxsackievirus group B1. Subsequently, individual PCRs were combined with multiplex PCR reactions. In general, multiplex real-time PCR assays showed comparable sensitivities and specificities with individual assays. A retrospective clinical evaluation showed increased pathogen detection in 29% of samples using conventional PCR methods. Prospective clinical evaluations were detected in 123 of the 227 (54%) total samples used in the multiplex real-time PCR analysis. The Norovirus genogroup II was found most frequently (23%), followed by Rotavirus (20%), the Hepatitis A virus (4.5%), Coxsackievirus (3.5%), and Norovirus genogroup I (2.6%). Internally controlled multiplex real-time PCR assays for the simultaneous detection of Rotavirus, Coxsackievirus group B, the Hepatitis A virus, and Norovirus genogroups I and II showed significant improvement in the diagnosis of viral gastroenteritis.     

单管半套式RT-PCR法检测贝类中轮状病毒的研究

轮状病毒(Rotavirus)是一种以贝类为载体的重要食源性病毒,目前来说,RT_PCR是贝类中轮状病毒最有效的检测方法,但通常由于病毒含量较低以及贝类中存在大量的PCR抑制物,致使将其运用于实际样本的检测时灵敏度仍较低。在实验室模拟自然环境,以人工播毒的方式使贝类富集轮状病毒,运用单管半套式RT_PCR法进行检测,并将单管半套式RT_PCR与ELISA、RT_PCR的检测灵敏度做了比较,表明单管半套式RT_PCR比ELISA的灵敏度高10 0 0倍,是传统RT_PCR的10倍,有时甚至10 0倍。另外,单管半套式RT_PCR法降低了实验过程中的内源性和外源性污染,使检测所需时间从6h缩短至4 5h ,大大改进和完善了食源性病毒检测方法。并同时以整只贝和仅以贝的消化道为样检测表明,以消化道为样时的病毒检出率和检测灵敏度较高。     

用环介导等温扩增技术快速检测粪便样本中的沙门菌

目的:建立快速检测粪便样本中的沙门菌的环介导等温扩增技术(LAMP),并着重在灵敏度和特异性方面对此方法进行评价。方法:利用LAMP针对沙门菌特定基因invA(靶基因)设计的6条特异引物,通过引物特异性识别特定基因invA上的8个独立区域来快速检测沙门菌;LAMP反应过程中会产生白色沉淀焦磷酸镁,故可以通过监测浊度来判定反应结果。结果:实时浊度仪监测反应结果表明,LAMP反应在60~65℃等温条件下50 min内完成;如果在反应前添加羟基萘酚兰,蓝色阳性结果很明显区别于紫色阴性结果;LAMP的最低检出限为6.97 pg/μL,PCR为69.7pg/μL,LAMP方法的检测灵敏度是PCR的10倍,且具有良好的特异性。结论:LAMP方法用于快速检测沙门菌,具有检测过程简单、实验装置简便、反应结果肉眼可辨、灵敏度高、特异性强的特点,对非沙门菌菌株的结果呈阴性,表明引物设计有很好的特异性。对粪便样本进行检测,发现具有同样的敏感性和特异性。这表明LAMP法是潜在的和有价值的在粪便样本中直接检测沙门菌的方法,具有快速、简便、低成本的特点。LAMP法适用于快速临床诊断。     

B组轮状病毒RT—PCR来源的cDNA探针核酸诊断方法的建立

近十年来,国内外学者相继报道在人以及牛、羊、猪、大鼠的腹泻粪样中检出Ｂ组轮状病毒〔１－３〕。洪涛等于１９８３年首次报道成人腹泻轮状病毒（ＡＤＲＶ）属于Ｂ组轮状病毒〔４〕。目前,轮状病毒Ｂ组已被公认为引起人及不同动物腹泻的流行病学的重要因素。由于Ｂ组轮...     

Comparison of different PCR methods for detection of Brucella spp. in human blood samples

For detection of Brucella species by PCR four DNA extraction methods and four targets were compared using pure culture of Brucella melitensis and the best conditions were applied in clinical samples. It was found that the MagNA Pure LC method was the most efficient and sensitive method showing a positive PCR reaction with DNA extracted from as low as 25 and 100 CFU suspended in one ml blood and one ml water, respectively. Detection of Brucella spp. by conventional PCR was investigated using four different targets. The results indicated that The B4-B5 amplification method was the most sensitive one as it could amplify DNA extracted from as a low as 25 and 100 CFU/ml suspended in one ml water and blood, respectively. Furthermore real-time PCR was able to detect Brucella using DNA extracted from as low as 50 CFU/ml blood and 15 CFU/ml water, respectively. The best and optimum detection conditions were applied to the clinical samples. Evaluation of conventional PCR assays on blood specimens confirmed 72% of the results obtained by conventional blood culture methods with a specificity of 95%, while serum samples had a sensitivity of 54% and specificity of 100%. Real-time PCR was generally found to be more sensitive and specific for detecting Brucella spp. in blood and serum samples compared to conventional PCR. The real-time PCR done on blood specimens confirmed 77.5% of the results obtained by conventional blood culture methods with specificity of 100%, while 60% of serum samples were found to be positive with specificity of 100%. These results suggest that serum and blood analysis by conventional and real time PCR is a convenient and safe method for rapid and accurate diagnosis of brucellosis.     

Development of a temperature sensor array chip and a chip-based real-time PCR machine for DNA amplification efficiency-based quantification

A temperature sensor array chip was developed to monitor the thermal cycling profiles of a polymerase chain reaction (PCR). DNA amplification efficiency of each cycle was estimated through temperature data to fit the stochastic model. A fluorescence detector system was constructed to detect the PCR amplifications of latter cycles, at which the fluorescence intensity passed the optical detection threshold. Through monitoring of both temperature and fluorescence, DNA amplification efficiency curve was completed for quantification. The F?rster resonance energy transfer (FRET) was employed to detect the measurements of the PCR product amount at the reaction endpoint. The chip-based, real-time PCR machine was constructed to perform the amplification efficiency curve-based quantification method. This novel method achieved the absolute quantification of the Hepatitis B virus (HBV) DNA using a single sample without the construction of the standard curve. The coefficient of variation (CV) of the 15 replicates inter assay experiments was less than 5.87%. Compared with the CV values obtained from the commercial machine in the range of 4.33-14.56%, it is noted that CV values of the prototype with respect to the samples of different initial concentration ranging from 10(7) to 10(3)copies/ml are almost equable.     

Rapid Detection of Oculopathogenic Adenovirus in Conjunctivitis

Ocular adenovirus (Ad) infections occur throughout the world in both sporadic and epidemic forms. Accurate laboratory diagnosis of Ad in conjunctival samples is always valuable. The present study was carried out to explore the presence of Ad as a causative agent in clinically suspected viral conjunctivitis and to compare the performance of conventional virus isolation on cell cultures, direct detection of Ad antigens in conjunctival cells by a direct fluorescence assay, Ad DNA detection by polymerase chain reaction (PCR), and specific IgM measurement by ELISA. Samples included scrapes from conjunctiva. Scrapes were subjected to study by direct immunofluorescence stain, culture on the Hep-2 cell line, and PCR for Ad detection. Blood samples were also taken and subjected to study for specific anti-Ad IgM determination. The culture for Ad was positive in 77.8%, direct antigen detection by fluorescent stain was positive in 72.2%, PCR was positive in 83.3%, and serology was positive in 88.9% of patients. Both determination of antibody IgM and PCR correctly identified a larger group of patients compared to cell culture. The most sensitive and specific method for diagnosis of Ad compared to culture was PCR (100%), followed by IgM detection (92.9%) then direct antigen detection by fluorescent stain (85.8%). From this study, we conclude that Ad is a common pathogen in sporadic cases of conjunctivitis. Screening of adenoviral conjunctivitis is possible by using specific IgM due to its high sensitivity. A confirmatory test can be done by PCR for diagnosis of Ad, as it is a rapid, specific, and accurate method.