Development of a highly sensitive nested-PCR method using a single closed tube for detection of Fusarium culmorum in cereal samples

AIMS: The aim of the study was to develop a sensitive detection method of Fusarium culmorum contamination in cereal samples. METHODS AND RESULTS: A nested-PCR method using a single closed tube was developed for the detection of F. culmorum in infected cereal samples. The concentrations of the first primer pair was diluted 10,000 times compared to the concentration used for the second primer pair. Differing annealing temperatures allowed both first and second polymerase chain reaction (PCR) reactions to be performed subsequently in the same closed tube. The detection limit was 5-50 fg of purified target DNA and allowed the detection of 1% infected seeds of wheat in a mixture with uninfected grains. CONCLUSIONS: F. culmorum can be specifically detected in cereal samples by the highly sensitive method of nested-PCR in a single closed tube. SIGNIFICANCE AND IMPACT OF THE STUDY: This work describes the detection of F. culmorum in cereal samples that is approximately 100 times more sensitive than previous PCR methods, involves low risk of cross contaminations between samples, low costs and reduced hands-on time as compared to standard nested-PCR protocols.     

Mitochondrial DNA common deletion is not associated with thyroid, breast and colorectal tumors in Turkish patients

Recently, efforts have been focused on mitochondrial DNA changes and their relation to human cancers. Among them, a 4977 bp deletion of mitochondrial DNA, named "common deletion", has been investigated in several types of tumors, with inconsistent results. In this study, we investigated the presence of the common deletion in tissues from 25 breast, 25 colorectal and 50 thyroid tumors and in the adjacent healthy tissues from Turkish patients. Samples from healthy volunteers were also evaluated for comparison. Two PCR-based methods were used for the detection of the common deletion. First, two pairs of primers were used to amplify wild-type and deleted mtDNA. Then, a highly sensitive nested-PCR was performed, to determine low amounts of deleted genomes. By the first method, wild-type mtDNAs were observed in all samples, but a deletion was observed in only six thyroid samples, by using the nested-PCR method. In conclusion, the mitochondrial common deletion was very rare in our study group and did not appear to be not related with cancer.     

Highly sensitive detection of Pseudomonas savastanoi pv. savastanoi in asymptomatic olive plants by nested-PCR in a single closed tube

A nested-polymerase chain reaction (PCR) has been set up to be performed in a single closed tube for the detection of Pseudomonas savastanoi pv. savastanoi. Nested-PCR coupled with dot-blot hybridization was able to detect up to one cell of the target per ml of olive extract, showing the greatest sensitivity compared with all previously reported detection assays. Validation of the developed procedure for diagnosis and epidemiological purposes was achieved by testing ca. 240 asymptomatic plant samples from olive trees. When performing the other previously reported techniques (bacterial isolation and single PCR), P. savastanoi was detected in 50 of the analyzed samples, while with the new developed nested-PCR assay, the bacterium was detected in 82 samples.     

双重套式PCR对不同取样胚胎性别鉴定灵敏度测试

目的:测定双重套式PCR微量扩增体系的灵敏度,为附植前遗传学诊断 (PGD)、家畜胚胎性别鉴定等提供技术保障。方法:以桑椹胚卵裂球为模板进行扩增,建立昆明白小鼠特异的SRY ZFX双重套式PCR性别鉴定体系。按卵裂球数量的不同分为五组,对应的卵裂球个数分别为 1、2、3、4、5及以上。根据有效扩增结果得出双重套式PCR的灵敏度。结果:第 1组 (卵裂球个数为 1 ),有效检出率为 85 % ( 34 /40 ),污染率为 7 5 % ( 3 /40 ),漏检率为 7 .5 % ( 3 /40 )。随着卵裂球个数的增多,有效检出率逐渐升高,而污染率和漏检率则呈逐渐下降趋势。第 5组 (卵裂球达到 5及以上 ),有效检出率达到 1 0 0 %,漏检率为 0。对于第 2、3、4、5组而言,有效检出率及漏检率各组间并无显著差异 (P >0 . 0 5 );而第一组与其它各组间,有效检出率及漏检率存在显著差异 (P <0 .0 5 )。结论:双重套式PCR可以有效扩增基因组DNA量约为 1 2pg的模板,且扩增片段为单拷贝片段。     

A Fluorescence Detection and Selection Device for Dna-Molecules Bound to Microspheres

Abstract

A device for the detection and selection of DNA-molecules bound to microspheres has been realized. It is based on a quartz chip with a capillary flowsystem, and confocal fluorescence detection of DNA. The device is to be used to identify and isolate single DNA-molecules for sequencing experiments.     

Identification of Plasmodium falciparum,P. vivax,P. ovale and P. malariae and detection of mixed infection in patients with imported malaria in Italy

The species-specific nested-PCR previously described by Snounou and others for detecting the four parasite species that cause human malaria is evaluated in the current study testing 230 blood samples. The results are compared with those obtained by microscopy and, for 101 samples out of 230, with those previously obtained by a genus-specific PCR based method (pg-PCR) followed by species-specific Southern-blot hybridization. All blood specimens were obtained from patients (127 foreigners and 103 Italians) with a suspect clinical diagnosis of imported malaria in Italy: 76 were positive by microscopy and 83 were positive by nested-PCR. The last method also revealed 10 double infections (8 foreigners and 2 Italians) which were not identified by microscopy or by pg-PCR with species-specific Southern-blot hybridization. Fifty-four out of 83 positive samples tested by nested-PCR were submitted to genomic sequence analysis, which confirmed the presence of DNA region portion encoding the 18S rRNA corresponding to the Plasmodium species identified by nested-PCR. These results demonstrate that the nested-PCR assay surpasses microscopy and pg-PCR with species-specific Southern-blot hybridization, both in sensitivity and in diagnostic accuracy. Moreover, it is quicker because it requires no further blotting or hybridization of PCR amplification products. This method also offers a clear advantage in the detection of mixed infections, which is important not only for successful medical treatment but also for the study of malaria epidemiology. Finally, our study also highlights the value of genomic sequence analysis for validating PCR results.     

A new TaqMan method for the identification of phytoplasmas associated with grapevine yellows by real-time PCR assay

Three real-time PCR systems for direct detection of phytoplasmas associated to Flavescence dorée (FD), Bois noir (BN) and aster yellows (AY) diseases were developed. TaqMan probes and primers were designed on the 16S ribosomal RNA sequences of phytoplasma genome. A further TaqMan assay, targeting a grapevine gene encoding for the chloroplast chaperonin 21, was developed in order to check the DNA quality and to verify the absence of PCR inhibition. A comparison between real-time PCR and conventional nested-PCR methods for phytoplasma detection was carried out on several reference samples from grapevine, periwinkle, other host plants and insect species. Detection of FD, BN and AY phytoplasma DNA on infected specimens was rapid, specific and reproducible. Sensitivity was as high as nested-PCR assay. The two procedures were then used on about 450 samples collected from grapevines showing yellows symptoms. The results showed that real-time PCR approach for phytodiagnostic purposes was more advantageous than nested-PCR method with regard to rapidity of the assay and reduced risk of sample cross contamination. These new protocols represent an improvement of existing analytical methods and could be used as a reliable diagnostic procedure in certification and control programs.     

Sensitivity of the nested-polymerase chain reaction (PCR) assay for Brugia malayi and significance of 'free' DNA in PCR-based assays

The blood filtration method was used as the gold standard to determine the detection level of simple blood-spot sampling and nested-polymerase chain reaction (PCR) for Brugia malayi. Of 100 samples, 48 were filtration-positive. Of these, 26 had microfilaria counts that were low enough (<1-29 microfilariae/ml) to accurately assess the limit of detection by nested-PCR. Nested-PCR consistently detected B. malayi DNA in samples with > or = 10 microfilariae/ml. Post-filtration, microfilaria-depleted, blood-spots from microfilaria-positive samples were screened by nested-PCR and B. malayi specific 'free' DNA was detected in 51.7% of these samples. There was no evidence for 'free' DNA in microfilaria-negative individuals from this endemic community.     

Nested-PCR检测恒河猴泡沫病毒

目的用nested-PCR检测恒河猴泡沫病毒SFV的前病毒形式。方法从SFV-1的pol区域选择两对引物分别对原代猴肾细胞（rhesus monkey kidney,RMK）及猴外周血淋巴细胞（peripheral blood lymphocytes,PBLs）进行体外扩增,扩增产物经1.0%的琼脂糖凝胶电泳,证实其特异性。阳性对照使用具有典型泡沫样病变的RMK377细胞株的前病毒DNA,阳性对照的PCR扩增产物经测序证实,阴性对照为恒河猴的SRV-1cDNA。结果nested-PCR能快速灵敏的直接从猴外周血淋巴细胞检出SFV的前病毒形式,与RMK细胞培养结果基本相一致。结论本实验所建立的检测SFV的nested-PCR法能快速准确的检测猴群中的SFV的带毒情况,对于提高实验猴的质量具有重要意义。     

PCR技术在附红细胞体感染模型中的应用

目的以猪附红细胞体(E.suis)为例,建立套式PCR方法,定性测定E.suis感染大鼠血液中DNA,检验套式PCR方法在其动物模型应用中的实用性和可操作性。方法用阳性血液样品分别经腹腔、皮下及肌肉感染大鼠,定期采血,进行常规血涂片染色,并从血液中分离纯化E.suis,提取DNA,进行套式PCR法扩增,凝胶电泳定性。结果套式PCR经扩增后得到了一长度为498bp的特异条带,测序鉴定确为目的片段;在整个实验过程中未见感染大鼠明显的临床症状。结论可以用大鼠建立猪附红细胞体动物感染动物模型;附红细胞体的感染方式有以下几种:腹腔、皮下及肌肉感染。在隐性感染的整个阶段,PCR方法依靠其高敏感性,作为猪附红细胞体感染大鼠模型检测指标之一,有其必要性和重要性。     

Nested-PCR and a New ELISA-Based NovaLisa Test Kit for Malaria Diagnosis in an Endemic Area of Thailand

Microscopy is considered as the gold standard for malaria diagnosis although its wide application is limited by the requirement of highly experienced microscopists. PCR and serological tests provide efficient diagnostic performance and have been applied for malaria diagnosis and research. The aim of this study was to investigate the diagnostic performance of nested PCR and a recently developed an ELISA-based new rapid diagnosis test (RDT), NovaLisa test kit, for diagnosis of malaria infection, using microscopic method as the gold standard. The performance of nested-PCR as a malaria diagnostic tool is excellent with respect to its high accuracy, sensitivity, specificity, and ability to discriminate Plasmodium species. The sensitivity and specificity of nested-PCR compared with the microscopic method for detection of Plasmodium falciparum, Plasmodium vivax, and P. falciparum/P. vivax mixed infection were 71.4 vs 100%, 100 vs 98.7%, and 100 vs 95.0%, respectively. The sensitivity and specificity of the ELISA-based NovaLisa test kit compared with the microscopic method for detection of Plasmodium genus were 89.0 vs 91.6%, respectively. NovaLisa test kit provided comparable diagnostic performance. Its relatively low cost, simplicity, and rapidity enables large scale field application.     

Design and application of a Staphylococcus-specific single strand conformation polymorphism-PCR analysis to monitor Staphylococcus populations diversity and dynamics during production of raw milk cheese

AIM: Development of a nested-PCR single strand conformation polymorphism (SSCP) assay targeting the 16S rRNA genes of the Staphylococcus genus, to monitor staphylococci in cheese. METHODS AND RESULTS: New primer sets to specifically amplify 16S rDNA of staphylococci were designed to be used in a nested-PCR SSCP assay. The method was efficient in discriminating the staphylococcal species most frequently found in cheese. It was validated by monitoring Staphylococcus populations in three productions of raw milk cheese. Analysis of milk samples revealed dominant SSCP peaks corresponding to Staphylococcus aureus, Staphylococcus equorum and Staphylococcus saprophyticus. After 12 h, the S. aureus peak became dominant. CONCLUSIONS: The combination of specific Staphylococcus nested-PCR and SSCP allows rapid and direct monitoring of staphylococci diversity and dynamics in milk and cheese. In the core of the cheeses studied, S. aureus may have ecological advantages against other Staphylococcus populations. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach is a promising tool to study the ecology of staphylococci in cheeses and in other food samples.     

rpoB-PCR amplified gene and temporal temperature gradient gel electrophoresis: a rapid tool to analyse bacterial strains representative of cold-smoked salmon microflora

AIM: To evaluate rpoB gene as a biomarker of microbial biodiversity associated to cold-smoked salmon by a novel nested-polymerase chain reaction/temporal temperature gradient gel electrophoresis (PCR/TTGE) technique applied on pure cultures of reference strains. METHODS AND RESULTS: DNA obtained from pure cultures of reference strains was used in a succession of a first PCR amplification of rpoB fragment with degenerated nonclamped primers and a nested-PCR with nondegenerated clamped primers. PCR products were then applied on a TTGE gel in order to analyse strains profile. High quantity of nested-PCR products were obtained for each tested strain and TTGE profiles showed a good separation between the different reference bacteria and an easy way to associate one band to one species. CONCLUSION: The nested-PCR/TTGE technique used in this study is a promising way of investigating bacterial community structure of cold-smoked salmon or other food matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of its single copy state leading to single band profiles in TTGE, rpoB constitute a good potential molecular marker for further development of cold-smoked salmon biodiversity analysis.     

A novel Real Time micro PCR based Point-of-Care device for Salmonella detection in human clinical samples

Our POC (Point of Care) device is intended to be a diagnostic tool for routine use in the clinical sector. The validation of the whole procedure, including bacterial genomic DNA isolation and the Real Time detection of Salmonella spp., was conducted on 29 clinical stool samples that had been diagnosed with Salmonella spp. by a routine culture technique. The entire process was achieved in a single microfluidic chip within 35 min. In comparison to the culture reference method that is used in the clinical laboratories, this new device performed well in regards to the analytical parameters of sensitivity, specificity and accuracy. Therefore, the POC device reported in this study proved to be very appropriate for the fully integrated analysis system. To the best of our knowledge, this is the first work to report the sample preparation and followed by Real Time PCR (Polymerase Chain Reaction) on a single 2.5 μl chamber chip for the detection of Salmonella spp. bacteria in stool samples.     

Sequence conservation of repeat 3 region of the gene coding for the 15 kDa polyprotein within human and simian Loa loa.

The human and simian strains of Loa loa microfilariae are morphologically identical even though their periodicities vary. When using primate models (Mandrillus sphinx) of human loaisis for vaccination trials, the absence of any ongoing simian L. loa infection must be demonstrated. Nested primers derived from a human strain of L. loa (targeted on the repeat 3 region of the gene encoding the 15 kDa polyprotein; 15r3) amplified at 366 bp sequence from simian L. loa genomic DNA and blood lysates from mandrills infected with simian L. loa. This nested-PCR assay has been tested on 12 amicrofilaremic (AMF) mandrills (without filarial microfilariae) and was positive in four mandrills. The nested-PCR product derived from simian L. loa genomic DNA and from three of four AMF mandrills has been sequenced. No difference was observed between the four sequences, which, in addition, were 99.18% identical to the 15r3 of human L. loa. Therefore, the 15r3 sequence is conserved within human and simian L. loa. These results suggest that the four PCR-positive mandrills without circulating microfilariae had occult simian L. loa infections. The study demonstrates the ability of a nested-PCR assay to identify animals naturally infected with simian L. loa.     

A nested-PCR assay for detection of Xylella fastidiosa in citrus plants and sharpshooter leafhoppers

AIMS: Detection of Xylella fastidiosa in citrus plants and insect vectors. METHODS AND RESULTS: Chelex 100 resin matrix was successfully standardized allowing a fast DNA extraction of X. fastidiosa. An amplicon of 500 bp was observed in samples of citrus leaf and citrus xylem extract, with and without symptoms of citrus variegated chlorosis, using PCR with a specific primer set indicating the presence of X. fastidiosa. The addition of insoluble acid-washed polyvinylpyrrolidone (PVPP) prior to DNA extraction of insect samples using Chelex 100 resin together with nested-PCR permitted the detection of X. fastidiosa within sharpshooter heads with great sensitivity. It was possible to detect up to two bacteria per reaction. From 250 sharpshooter samples comprising four species (Dilobopterus costalimai, Oncometopia facialis, Bucephalogonia xanthopis and Acrogonia sp.), 87 individuals showed positive results for X. fastidiosa in a nested-PCR assay. CONCLUSIONS: The use of Chelex 100 resin allowed a fast and efficient DNA extraction to be used in the detection of X. fastidiosa in citrus plants and insect vectors by PCR and nested-PCR assays, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: The employment of efficient and sensitive methods to detect X. fastidiosa in citrus plants and insect vectors will greatly assist epidemiological studies.     

从粪便样品中检测动物冠状病毒的分子技术研究

目的 感染冠状病毒的动物向环境排毒主要是通过粪便,建立直接从粪便样品对动物冠状病毒进行检测的分子技术具有重要的公共卫生学意义。方法 通过计算机模拟和实验方法对已报道的2对针对冠状病毒pol基因的通用引物的通用性进行了验证。不经传统的病毒分离．直接从环境样品中提取病毒RNA,通过一步法RT-PCR进行检测,并通过分子杂交和Nested PCR扩增结合TaqMan探针实时荧光检测的PCR技术．提高对冠状病毒检测的灵敏度和准确度,并对猪、禽冠状病毒感染的临床样品进行分析检测。结果 2对引物可以覆盖所有已知的冠状病毒,包括SARS,RT-PCR产物通过测序可以确定冠状病毒种类;实时荧光定量NestedPCR有很高的灵敏度,可以灵敏地检出所有供试的阳性样品,而荧光增量实时监测可以排除凝胶电泳检查的假阳性。结论 该研究为从环境中普查和鉴定冠状病毒提供了可靠的技术方法。