Immunoenzymatic Detection of PCR Products for the Identification of Phytoplasmas in Plants

Polymerase chain reaction (PCR, with universal and specific primers designed on rRNA genes) provides a rapid, reliable method of diagnosing phytoplasmas (formerly mycoplasma-like organisms) in plants. However, to attain a better identification of these prokaryotes, it is often necessary to digest the PCR products with restriction endonucleases or to hybridize them with specific probes. The present study compared routine procedures for detecting PCR products against a new system, PCRELISA (Boehringer Mannheim), which enables immunoenzymatic detection of PCR products. The results show that this new system provides fast and highly sensitive detection of several phytoplasmas associated with certain trees and shrubs. Optimization of all parameters involved in the PCR-ELISA procedure and its advantages are reported and discussed.     

Rapid detection of Listeria monocytogenes by PCR-ELISA

A rapid detection system specific for Listeria monocytogenes based upon the polymerase chain reaction was developed. The specificity of the primers and the probe annealing to the coding region of the mpl gene proved positive with the DNA from a total of 103 L. monocytogenes strains, while DNA from another 73 Listeria and non-Listeria strains tested negative. To facilitate detection with large numbers of samples, a microtitre plate assay was established with biotinylated probes. Use of a standard DNA prevented false-negative results when used as an internal amplification control in the PCR-ELISA. As the described method required approximately 5-6 h to be completed it may prove useful in the detection of L. monocytogenes in food.     

金黄色葡萄球菌反向线性杂交探针检测方法的建立

目的建立一种检测金黄色葡萄球菌的简单、快速、灵敏、准确的方法。方法根据金黄色葡萄球菌的耐热核酸酶nuc基因,设计一对通用引物及两条特异性探针,用生物素标记通用引物的5＇端,将两条特异性探针固定于硝酸纤维膜上,使PCR产物与探针杂交。结果建立的反向线性杂交探针方法,其检测限为2 ng/μL,检测特异性和准确性均为100%。结论建立的反向线性杂交检测方法具有较高的敏感性和特异性,可用于实验动物金黄色葡萄球菌的快速检测。     

Qualitative PCR-ELISA protocol for the detection and typing of viral genomes

PCR is an established technique providing rapid and highly productive amplification of specific DNA sequences. The demand for equally rapid, sensitive and objective methods to achieve detection of PCR products has led to the coupling of PCR with ELISA. PCR-ELISA involves direct incorporation of labeled nucleotides in amplicons during PCR-amplification, their hybridization to specific probes and hybrid capture-immunoassay in microtiter wells. PCR-ELISA is performed in 1 d and is very flexible, with the ability to process simultaneously up to 96 or 384 samples. This technique is potentially automatable and does not require expensive equipment, and thus can be fundamental in laboratories without access to a real-time PCR thermocycler. PCR-ELISA has mainly been used to detect infectious agents, including viruses, bacteria, protozoa and fungi. A PCR-ELISA protocol for the qualitative detection of papillomavirus genomes and simultaneous typing of different genotypes are detailed here as an example of the technique.     

PCR-ELISAs for the detection of Campylobacter jejuni and Campylobacter coli in poultry samples

Campylobacter species, primarily Campylobacter jejuni and Campylobacter coli, are regarded as a major cause of human gastrointestinal disease, commonly acquired by eating undercooked chicken. We describe a PCR-ELISA for the detection of Campylobacter species and the discrimination of C. jejuni and C. coli in poultry samples. The PCR assay targets the 16S/23S ribosomal RNA intergenic spacer region of Campylobacter species with DNA oligonucleotide probes designed for the specific detection of C. jejuni, C. coli, and Campylobacter species immobilized on Nucleo-Link wells and hybridized to PCR products modified with a 5' biotin moiety. The limit of detection of the PCR-ELISA was 100-300 fg (40-120 bacterial cells) for C. jejuni and C. coli with their respective species-specific oligonucleotide probes and 10 fg (4 bacterial cells) with the Campylobacter genus-specific probe. Testing of poultry samples, which were presumptive positive for Campylobacter following culture on the Malthus V analyzer, with the PCR-ELISA determined Campylobacter to be present in 100% of samples (n = 40) with mixed cultures of C. jejuni/C. coli in 55%. The PCR-ELISA when combined with culture pre-enrichment is able to detect the presence of Campylobacter and definitively identify C. jejuni and C. coli in culture-enriched poultry meat samples.     

Real time PCR hybridization for the rapid and specific identification of Francisella tularensis

Tularemia is highly infectious and fatal zoonotic disease caused by Gram negative bacteria Francisella tularensis. The necessity to undergo medical treatment in early phase of illness in humans and possibility of making use of bacterial aerosol by terrorists in an attack create an urgent need to implement a rapid and effective method which enables to identify the agent. In our study two primers FopA F/R and hybridization probes FopA S1/S2 designed from fopA gene sequence, were tested for their potential applicability to identify F. tularensis. In this research 50 strains of F. tularensis were used and the test gave positive results. Reaction specificity was confirmed by using of non-Francisella tularensis bacterial species. The results obtained in the real-time PCR reaction with primers Tul4 F/R and hybridization probes Tul4 S1/S2, designed from tul4 gene, were comparable to the results from previous experiment with fopA - primers set. Investigation of fopA and tul4 primers and hybridization probes properties revealed characteristic Tm (melting temperature) value of the products--61 degrees C and 60 degrees C, respectively. Detection sensitivity was remarkably higher when fopA primers set was used 1 fg/microl, and for tul4 primers set, minimal detectable concentration is 10 fg/microl.     

Competitive PCR-ELISA protocols for the quantitative and the standardized detection of viral genomes

Competitive PCR-ELISA combines competitive PCR with an ELISA to allow quantitative detection of PCR products. It is based on the inclusion of an internal standard competitor molecule that is designed to differ from the target by a short sequence of nucleotides. Once such a competitor molecule has been designed and constructed, target and competitor sequences are concurrently PCR-amplified, before hybridization to two different specific probes and determination of their respective OD values by ELISA. The target can be quantified in relation to a titration curve of different dilutions of the competitor. The competitor can alternatively be used at a unique optimal concentration to allow for standardized detection of the target sequence. PCR-ELISA can be performed in 1 d in laboratories without access to a real-time PCR thermocycler. This technique is applied in diagnostics to monitor the course of infections and drug efficacy. Competitive PCR-ELISA protocols for the quantitative and for the standardized detection of parvovirus B19 are detailed here as an example of the technique.     

Rapid detection of Campylobacter coli,C. jejuni,and Salmonella enterica on poultry carcasses by using PCR-enzyme-linked immunosorbent assay

Contamination of retail poultry by Campylobacter spp. and Salmonella enterica is a significant source of human diarrheal disease. Isolation and identification of these microorganisms require a series of biochemical and serological tests. In this study, Campylobacter ceuE and Salmonella invA genes were used to design probes in PCR-enzyme-linked immunosorbent assay (ELISA), as an alternative to conventional bacteriological methodology, for the rapid detection of Campylobacter jejuni, Campylobacter coli, and S. enterica from poultry samples. With PCR-ELISA (40 cycles), the detection limits for Salmonella and Campylobacter were 2 x 10(2) and 4 x 10(1) CFU/ml, respectively. ELISA increased the sensitivity of the conventional PCR method by 100- to 1,000-fold. DNA was extracted from carcass rinses and tetrathionate enrichments and used in PCR-ELISA for the detection of Campylobacter and S. enterica, respectively. With PCR-ELISA, Salmonella was detected in 20 of 120 (17%) chicken carcass rinses examined, without the inclusion of an enrichment step. Significant correlation was observed between PCR-ELISA and cultural methods (kappa = 0.83; chi-square test, P < 0.001) with only one false negative (1.67%) and four false positives (6.67%) when PCR-ELISA was used to screen 60 tetrathionate enrichment cultures for SALMONELLA: With PCR-ELISA, we observed a positive correlation between the ELISA absorbance (optical density at 405 nm) and the campylobacter cell number in carcass rinse, as determined by standard culture methods. Overall, PCR-ELISA is a rapid and cost-effective approach for the detection and enumeration of Salmonella and Campylobacter bacteria on poultry.     

双重实时荧光PCR法检测食品和饲料中的鸡源性成分

根据鸡线粒体DNA细胞色素b基因序列和内参照基因PUC18质粒基因序列设计特异性引物和以不同荧光素标记的TaqMan探针。通过对反应条件的优化筛选,建立能同时扩增鸡源性成分和内参照基因成分的双重实时荧光PCR检测方法。设置内参照反应是为了监控反应体系中是否含有PCR反应的抑制物,避免出现假阴性结果。分别以鸡、鸭、鹅、火鸡、牛、羊、猪、鱼、兔、驴、鹿、狗、马、鸽子、大豆、玉米、小麦、大米、马铃薯及番茄的线粒体DNA作为模板进行特异性试验,结果表明该方法仅能特异性扩增鸡源性成分,而对其它物种未见有效扩增。通过灵敏度测试,该方法检出限达0.01%。所制定的方法特异性高,灵敏度好,可以作为食品和饲料中鸡源性成分的高效检测方法。     

Rapid screening of libraries with radiolabeled DNA sequences generated by PCR using highly degenerate oligonucleotide mixtures.

The PCR technique can use protein-derived oligonucleotide sequences as primers to develop probes for screening recombinant libraries. Here we report a method with highly degenerate mixtures of oligonucleotides as primers for the PCR that eliminates the need to identify or isolate the DNA sequences derived by PCR. The method uses the pool of PCR-generated DNA sequences radiolabeled during the extension reaction as a probe, combined with highly stringent hybridization and wash conditions that permit only homologous sequences to hybridize and therefore target desired clones. This technique was used successfully to clone the receptor for tumor necrosis factor.     

Evaluation of the specificity of Salmonella PCR primers using various intestinal bacterial species

AIMS: Nine sets of PCR primers targeting Salmonella were evaluated for their specificity with pure cultures of intestinal-associated bacteria prior to their application to Salmonella detection in faecal samples. METHODS AND RESULTS: Gene targets of PCR primers included: 16S rDNA, a Salmonella pathogenicity island I virulence gene, Salmonella enterotoxin gene (stn), invA gene, Fur-regulated gene, histidine transport operon, junction between SipB and SipC virulence genes, Salmonella-specific repetitive DNA fragment, and multiplex targeting invA gene and spvC gene of the virulence plasmid. Fifty-two Salmonella strains were used to determine sensitivity; five strains from related genera and 45 intestinal bacteria were used to evaluate specificity. All primers amplified DNA from Salmonella strains, although two primer sets failed to amplify Salmonella DNA from either Salmonella bongori (hilA) or subgroups VI or VII (16S rDNA). There was no detected amplification of DNA from related bacterial genera with any of nine PCR assays. Six of the PCR assays amplified DNA for some intestinal bacteria. CONCLUSIONS: Only three primer pairs were determined to be suitable for application of PCR amplification of Salmonella in faecal samples - 16S rDNA, stn and histidine transport operon. We are currently evaluating their sensitivity of detection of Salmonella in faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the importance of internal lab validation of PCR primers prior to application to the type of samples of interest. Information from this evaluation can be applied in other labs to facilitate choosing Salmonella PCR primers.     

Quantification of Leishmania infantum parasites in tissue biopsies by real-time polymerase chain reaction and polymerase chain reaction-enzyme-linked immunosorbent assay

Most of the experimental studies of Leishmania spp. infection require the determination of the parasite load in different tissues. Quantification of parasites by microscopy is not very sensitive and is time consuming, whereas culture microtitrations remain laborious and can be jeopardized by microbial contamination. The aim of this study was to quantify Leishmania infantum parasites by real-time polymerase chain reaction (PCR) using specific DNA TaqMan probes and to compare the efficacy of detection of this technique with a PCR-enzyme-linked immunosorbent assay (ELISA). For this purpose, spleen and liver samples from L. infantum-infected mice were collected during a 3-mo longitudinal study and analyzed by both methods. PCR-ELISA failed to quantify Leishmania spp. DNA in samples with very low or very high numbers of parasites. Real-time PCR was more sensitive than PCR-ELISA, detecting down to a single parasite, and enabled the parasite quantification over a wide, 5-log range. In summary, this study developed a method for absolute quantification of L. infantum parasites in infected organs using real-time TaqMan PCR.     

多重实时荧光PCR检测牛、山羊和绵羊源性成分

根据牛、山羊和绵羊线粒体细胞色素b基因序列, 设计特异性引物和以不同荧光素标记的Taqman探针。通过对PCR反应体系和反应条件的优化筛选, 建立能同时鉴别牛、山羊和绵羊源性成分的多重实时荧光PCR方法。采用本文方法与国标GB/T 20190-2006方法分别对17种不同源性动物DNA和200份不同来源样品DNA进行牛羊源性成分检测, 数据显示两者检测结果符合率达100%, 特异性相当。与国标方法相比, 本试验方法不需电泳、酶切和测序, 即可在一个PCR反应中同时鉴别检测牛、山羊和绵羊3种源性成分, 检测效率提高近3倍; 灵敏度更高, 比国标方法灵敏10倍; 适用性更广, 除了饲料, 还适用于肉品、奶品、生皮和动物油脂等动物产品的牛羊源性成分检测。     

The impact of genetic diversity in protozoa on molecular diagnostics

Detection of intestinal parasitic protists, commonly referred to as 'intestinal protozoa,' by PCR is increasingly used not only for identification or confirmation but also as a first-line diagnostic tool. Apart from the ability to sample correctly and extract parasite DNA directly from faeces, primer and probe specificity and sensitivity affect predictive values and hence the utility of diagnostic assays. Molecular characterization of intestinal protists is necessary to design primers and probes because this is the basic material for current and future improved diagnostic PCRs for either detecting all genetic variants or specifically differentiating among such variants. As an example, this paper highlights the existence of interspecific and intraspecific genetic diversity among intestinal, unicellular parasites and its implications for nucleic acid-based diagnostic assays.     

基因芯片技术检测3种肠道病原微生物方法的建立

目的:建立一种运用多重PCR和基因芯片技术检测和鉴定伤寒沙门氏菌、痢疾杆菌和单核细胞增生利斯特菌的方法。方法:分别选取伤寒沙门氏菌染色体ViaB区域中编码调控Vi抗原表达的基因(vipR)、痢疾杆菌编码侵袭质粒抗原H基因(ipaH)和单核细胞增生利斯特菌溶血素基因(hlyA)设计引物和探针,探针3'端进行氨基修饰,下游引物标记荧光素Cy3。在优化的PCR和杂交反应条件下,进行三重PCR扩增,产物与包括3种致病菌特异性探针的基因芯片杂交。在评价基因芯片的特异性和灵敏度之后,对临床样本进行检测。结果:只有3种目的致病菌的PCR产物在相应探针位置出现特异性信号,其他阴性细菌均无信号出现;3种致病菌的检测灵敏度均可达到103CFU/mL;检测30例临床样本的结果与常规细菌学培养结果一致。结论:所建立的可同时检测伤寒沙门氏菌、痢疾杆菌和单核细胞增生利斯特菌的基因芯片方法快速、准确,特异性高,重复性好,为3种肠道致病菌的快速检测和鉴定提供了新方法和新思路。     

A new diagnostic system for ultra-sensitive and specific detection and quantification of Candidatus Liberibacter asiaticus, the bacterium associated with citrus Huanglongbing

An ultra-sensitive and quantitative diagnostic system by combining nested PCR and TaqMan® PCR in a single tube was developed for detection of “Candidatus Liberibacter asiaticus”. The procedure involves two PCR steps using the species-specific outer and inner primer pairs. Different annealing temperatures allow both the first and the second rounds of PCR to be performed sequentially in the same closed tube. The first PCR with outer primers was performed at a higher annealing temperature and with limited amount of primers to prevent interference with the inner primers during the second round of PCR. The specificity of the dual primer TaqMan® is high because the fluorescent signal can only be generated from the TaqMan® probes that are homologous to the product amplified by the outer and inner primers. This new detection system can reliably detect as few as single copies of target DNA. The sensitivity of the dual primer system is comparable to the conventional two-tube nested PCR, but it eliminates the potential risk of cross contamination commonly associated with conventional nested PCR. This one-tube dual primer TaqMan® PCR method is gel-free with reduced handling time and is cost effective. At the same time, this system provides significantly increased sensitivity, improved reliability and high through-put capability suitable for routine, large scale diagnoses of clinical plant tissue and insect samples. The technique described here is generic and can be applied to the detection of other plant pathogenic bacteria.     

Distribution of bifidobacterial species in human intestinal microflora examined with 16S rRNA-gene-targeted species-specific primers.

In order to clarify the distribution of bifidobacterial species in the human intestinal tract, a 16S rRNA-gene-targeted species-specific PCR technique was developed and used with DNAs extracted from fecal samples obtained from 48 healthy adults and 27 breast-fed infants. To cover all of the bifidobacterial species that have been isolated from and identified in the human intestinal tract, species-specific primers for Bifidobacterium longum, B. infantis, B. dentium, and B. gallicum were developed and used with primers for B. adolescentis, B. angulatum, B. bifidum, B. breve, and the B. catenulatum group (B. catenulatum and B. pseudocatenulatum) that were developed in a previous study (T. Matsuki, K. Watanabe, R. Tanaka, and H. Oyaizu, FEMS Microbiol. Lett. 167:113-121, 1998). The specificity of the nine primers was confirmed by PCR, and the species-specific PCR method was found to be a useful means for identifying Bifidobacterium strains isolated from human feces. The results of an examination of bifidobacterial species distribution showed that the B. catenulatum group was the most commonly found taxon (detected in 44 of 48 samples [92%]), followed by B. longum and B. adolescentis, in the adult intestinal bifidobacterial flora and that B. breve, B. infantis, and B. longum were frequently found in the intestinal tracts of infants. The present study demonstrated that qualitative detection of the bifidobacterial species present in human feces can be accomplished rapidly and accurately.     

A PCR-ELISA for the identification of cyathostomin fourth-stage larvae from clinical cases of larval cyathostominosis

We report the use of six oligoprobes designed from intergenic spacer region sequences to identify fourth-stage larvae (L4) of the tribe Cyathostominae. Oligoprobes were designed for identification of the following species: Cylicocyclus ashworthi, Cylicocyclus nassatus, Cylicocyclus insigne, Cyathostomum catinatum, Cylicostephanus goldi, and Cylicostephanus longibursatus. A seventh probe was designed as a positive control to identify all these members of the Cyathostominae. The intergenic spacer region was amplified by PCR using conserved primers. Initially, three oligoprobes were used in Southern blot analysis. To facilitate high-throughput identification, these and a further four oligoprobes were developed for use in a PCR-ELISA. All probes were validated for their ability to detect cyathostomin PCR products in the PCR-ELISA, using DNA from morphologically identified adult parasites. Initially, 712 L4 were isolated from the diarrhoeic faeces from horses (n=17) with clinical larval cyathostominosis. PCR products from 522 of these L4 were subjected to analysis, with 413 L4 being identified as one of the aforementioned species. With reference to individual species analysis, 28.5% of the 522 L4 were identified as C. longibursatus, 25.7% as C. nassatus, 15.9% as C. ashworthi, 7.3% as C. goldi and 1.7% as C. catinatum. No L4 were identified as being C. insigne species. When L4 within faeces from individual horses were compared, no sample was found to comprise parasites of one species. The least number of species identified in a single sample was two. This study suggests that clinical larval cyathostominosis is predominantly caused by mixed-species infections.     

Quantitative and qualitative analysis of amplified DNA sequences by a competitive hybridization assay.

The presence of allelic sequence variations in DNA fragments can be easily detected by measuring the extent of DNA strand exchange between test double-stranded PCR products (target) and labeled standard double-stranded PCR products (probe). Under selected hybridization conditions, sequences identical to the probe decreased the formation of double-labeled hybrid, whereas differing sequences were not efficient enough to compete with the regeneration of the probe. A single base substitution in the target DNA increased the percentage of remaining double-labeled probe. A general procedure involving denaturation and hybridization in solution under different temperature conditions or using different probes enabled sequence identification. The degree of regeneration of double-labeled probe was determined using a bioluminescent assay. We evaluated the specificity of this method with two probes (108 and 131 bp) and several targets with different base substitutions.