Modification and optimization of PCR methods for detection of MIE region from human herpesvirus 5

Human herpesvirus 5 (HHV-5, formerly known as CMV) is a beta-herpesvirus widely spread within a population. Thus, HHV-5 infections are a serious matter of concern in a group of immunocompromised patients. The goal of the study was modification and optimization of conventional PCR method developed for the detection of HHV-5 DNA to the real-time variant (RTmPCR) and determination of analytical resolution of the modified methods. Thirty plasma samples were tested for the presence of HHV-5 DNA using the LightCycler system with two different methods--one with SYBR Green I fluorochrome method and second one using TaqMan fluorescent probes and a qualitative in-house gel-stained PCR assay using primers that amplify part of HHV-5 MIE gene. The analytical sensitivity of real-time PCR assay was tested using serial dilutions of HHV-5 DNA in range between 10(0) and 10(-6). For comparison typical end-point detected PCR for cytomegalovirus detection with the same DNA dilutions was made. The sensitivity of novel method was about 100-fold higher than older one. Both LightCycler assays detected HHV-5 DNA in 27 samples, also which were negative by the gel-stained PCR. Analysis of the available clinical and serological data associated with these samples suggested that the real-time results in all of these cases were true positive. The conclusion is that real-time PCR methods are more sensitive than the conventional PCR used in this study. The additional sensitivity was valuable for detection of patients with low-copy viremia. The high level of sensitivity, specificity, accuracy, and rapidity provided by the LightCycler instrument are favorable for the use of this system in the detection of HHV-5 DNA in clinical specimens.     

Quantification of genetically modified soybeans using a combination of a capillary-type real-time PCR system and a plasmid reference standard

Because the labeling of grains and feed- and foodstuffs is mandatory if the genetically modified organism (GMO) content exceeds a certain level of approved genetically modified varieties in many countries, there is a need for a rapid and useful method of GMO quantification in food samples. In this study, a rapid detection system was developed for Roundup Ready Soybean (RRS) quantification using a combination of a capillary-type real-time PCR system, a LightCycler real-time PCR system, and plasmid DNA as the reference standard. In addition, we showed for the first time that the plasmid and genomic DNA should be similar in the established detection system because the PCR efficiencies of using plasmid DNA and using genomic DNA were not significantly different. The conversion factor (Cf) to calculate RRS content (%) was further determined from the average value analyzed in three laboratories. The accuracy and reproducibility of this system for RRS quantification at a level of 5.0% were within a range from 4.46 to 5.07% for RRS content and within a range from 2.0% to 7.0% for the relative standard deviation (RSD) value, respectively. This system rapidly monitored the labeling system and had allowable levels of accuracy and precision.     

Rapid detection of the 46C --> T polymorphism in the factor XII gene, a novel genetic risk factor for thrombosis, by melting peak analysis using fluorescence hybridization probes

Factor XII (FXII) level is an important intermediate phenotype associated with thrombotic disease. The 46C --> T transition in the exon 1 of the Factor XII (F12) gene is a significant, prevalent, and independent genetic risk factor for thrombotic disease. It is also associated with interindividual variation of plasma FXII zymogen levels. The aims of this study were to develop a rapid, reproducible, and easy method for 46C --> T genotyping and to compare its reliability with the classical endonuclease digestion methodology. DNA samples from 100 subjects were genotyped for the 46C --> T transition using the classical endonuclease digestion method with Sfna I. The genotypes of three of them (each with a different 46C R T genotype) were confirmed by direct sequencing analysis. We then set out to construct a LightCycler PCR protocol to detect the 46C --> T polymorphism. This protocol was designed to combine a rapid-cycle polymerase chain reaction (PCR) with an allele-specific fluorescent probe melting for mutation detection. In the three sequenced samples, as well as in the remaining 97, the LightCycler PCR procedure unambiguously resulted in the same genotype previously observed by sequencing and endonuclease digestion. Characteristic fluorescent curves were obtained for each genotype; the first derivative of these curves had a maximum at an apparent hybridization temperature (Tm) that was specific for each probe/allele duplex. The whole process took less than 40 min. Thus, if this method is used with a rapid DNA extraction, the genotypes would be obtained within 60 min after receiving a blood sample. In conclusion, the technique presented allows for easy, reliable, and rapid detection of this polymorphism, and is suitable for typing both small and large numbers of DNA samples.     

Detection of Coxiella burnetii in complex matrices by using multiplex quantitative PCR during a major Q fever outbreak in The Netherlands

Q fever, caused by Coxiella burnetii, is a zoonosis with a worldwide distribution. A large rural area in the southeast of the Netherlands was heavily affected by Q fever between 2007 and 2009. This initiated the development of a robust and internally controlled multiplex quantitative PCR (qPCR) assay for the detection of C. burnetii DNA in veterinary and environmental matrices on suspected Q fever-affected farms. The qPCR detects three C. burnetii targets (icd, com1, and IS1111) and one Bacillus thuringiensis internal control target (cry1b). Bacillus thuringiensis spores were added to samples to control both DNA extraction and PCR amplification. The performance of the qPCR assay was investigated and showed a high efficiency; a limit of detection of 13.0, 10.6, and 10.4 copies per reaction for the targets icd, com1, and IS1111, respectively; and no cross-reactivity with the nontarget organisms tested. Screening for C. burnetii DNA on 29 suspected Q fever-affected farms during the Q fever epidemic in 2008 showed that swabs from dust-accumulating surfaces contained higher levels of C. burnetii DNA than vaginal swabs from goats or sheep. PCR inhibition by coextracted substances was observed in some environmental samples, and 10- or 100-fold dilutions of samples were sufficient to obtain interpretable signals for both the C. burnetii targets and the internal control. The inclusion of an internal control target and three C. burnetii targets in one multiplex qPCR assay showed that complex veterinary and environmental matrices can be screened reliably for the presence of C. burnetii DNA during an outbreak.     

A rapid PCR-based method for identification of four important Candida species

The rapid detection and identification of Candida species in clinical laboratories are extremely important for the management of patients with hematogenous candidosis. Currently available culture and biochemical methods for detection and identification of Candida species are time-consuming. This study describes the use of a simple and rapid PCR method using species-specific oligonucleotides for the detection of clinical isolates of Candida species. These species-specific oligonucleotides are complementary to unique sequences within the intergenic transcribed spacer 2, located in between the 5.8S and 28S ribosomal DNA, and generated DNA fragments by both the conventional and hemi-nested PCR reactions. Conventional PCR produced a single DNA fragment of variable size in all isolates, while the hemi-nested PCR produced two discrete DNA fragments, both with the expected sizes of 111bp/57bp (C. albicans), 84bp/42bp (C. glabrata), 94bp/45bp (C. krusei) and 95bp/49bp (C. parapsilosis). In conclusion, the PCR-based method described in this study is fast and specific for the identification of clinically important Candida species.     

胶体金渗滤法检测贝氏柯克斯体的研究

本文建立一种快速、敏感、适于基层应用的贝氏柯克斯体（俗称Ｑ热立克次体）的检测方法。将Ｑ热立克次体多克隆抗体点于硝酸膜上,用以捕获待检标本中的Ｑ热立克次体抗原,通过胶体金标记的鼠抗Ｑ热立克次体单克隆抗体直接显色,阳性者出现红色斑点。结果表明,用该法检测Ｑ热立克次休实验感染豚鼠血液,小鼠肝或脾,蜱血淋巴等标本取得了较满意的结果,整个过程仅需５－６分钟。与其他病原体无交叉反应,敏感度不低于５０ｎｇ立克次     

Statistical diagnostics emerging from external quality control of real-time PCR

Besides the application of conventional qualitative PCR as a valuable tool to enrich or identify specific sequences of nucleic acids, a new revolutionary technique for quantitative PCR determination has been introduced recently. It is based on real-time detection of PCR products revealed as a homogeneous accumulating signal generated by specific dyes. However, as far as we know, the influence of the variability of this technique on the reliability of the quantitative assay has not been thoroughly investigated. A national program of external quality assurance (EQA) for real-time PCR determination involving 42 Italian laboratories has been developed to assess the analytical performance of real-time PCR procedures. Participants were asked to perform a conventional experiment based on the use of an external reference curve (standard curve) for real-time detection of three cDNA samples with different concentrations of a specific target. In this paper the main analytical features of the standard curve have been investigated in an attempt to produce statistical diagnostics emerging from external quality control. Specific control charts were drawn to help biochemists take technical decisions aimed at improving the performance of their laboratories. Overall, our results indicated a subset of seven laboratories whose performance appeared to be markedly outside the limits for at least one of the standard curve features investigated. Our findings suggest the usefulness of the approach presented here for monitoring the heterogeneity of results produced by different laboratories and for selecting those laboratories that need technical advice on their performance.     

Rapid detection of diarrheagenic E. coli by real-time PCR

Enterovirulent Escherichia coli are among the most important causes of acute diarrhea in developing as well as in developed countries. We have adapted classical PCR to detect these organisms in stool specimens to real-time PCR using the LightCycler (LC) SYBR Green format followed by melting curve analysis. With only two different cycling protocols we could detect enteropathogenic E. coli (EPEC) and verocytotoxin-producing E. coli (VTEC) (duplex assay for both Verotoxin 1 (VT1) and Verotoxin 2 (VT2)) in one run and enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC) and enterotoxigenic E. coli (ETEC) (duplex assay detecting both heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT)) in another run. Using serial dilutions of control strains, the LC proved to be clearly more sensitive than conventional PCR for five out of seven investigated targets: VTEC (VT1 and VT2), ETEC (ST and LT) and EIEC. For EPEC and EAEC, LC and conventional PCR had identical sensitivities. With stool samples, we found an optimal agreement between LC-PCR and the conventional PCR when samples were tested in a 1:10 dilution. Only one specimen was discrepant, being repetitively positive for VT by LightCycler but not by conventional PCR. Given the significantly higher sensitivity of the LC-PCR for the VT target (up to a 10(-4) dilution factor by melting curve analysis and up to a 10(-6) dilution factor following gel electrophoresis), this is probably a false negative result by conventional PCR. We conclude that LightCycler PCR is more rapid, easier than and at least as sensitive as our conventional PCR for the detection of enterovirulent E. coli in stool specimens after culture on MacConkey.     

Detecting and measuring small numbers of viable Coxiella burnetii

Coxiella burnetii is an acidophilic, intracellular bacterium that causes the human disease Q fever. In some studies, it is important to distinguish between viable and nonviable C.?burnetii. We compared four methods for detecting and measuring viable C.?burnetii in biological samples as follows: growth in two different cell culture lines, infection of severe combined immunodeficient (SCID) mice (leading to death) and infection of SCID mice with detection of C.?burnetii in their spleen (after euthanasia at day 50 postinfection). Two isolates of C.?burnetii were used ('Henzerling' and 'Arandale'). Our in-house qPCR assay for C.?burnetii DNA was used as a control. SCID mouse inoculation was more sensitive than cell culture. The assay that detected C.?burnetii in SCID mouse spleens was slightly more sensitive than SCID mice deaths alone. Approximately one viable C.?burnetii cell could be detected by this method, making it suitable for determining the viability of C.?burnetii in a sample.     

PCR法对鸡卵黄中Coxiella burnetii菌的测定

[目的]通过用定量PCR加巢式PCR方法,提高了对Coxiella burnetii (C.b)CoMl基因的检出率;通过对鸡卵中病原微生物Coxiella burnetii的基因检测,明确鸡卵的食品安全性;并对明确Coxiella burnetii的流行病学有重要意义.[方法]提取鸡卵DNA,用定量PCR加巢式PCR方法检测上述基因,并对PCR产物进行测序分析,通过间接免疫荧光法观察鸡血白细胞中的微生物.[结果]用定量PCR加巢式PCR方法可检出4个以上的Coxiella burnetii Coml基因,用此方法可测出鸡卵中Coxiella burnetii Coml基因达104-106个,阳性率为5％-22％;对阳性鸡卵Coml基因PCR产物的测序结果显示有变异菌株的存在;免疫荧光法可见鸡卵中含有该微生物.[结论]由此认为鸡卵中存在病原微生物Coxiella burnetii,可能是Q热传染源.     

Detection of polymorphisms at exons 3 (Tyr113-->His) and 4 (His139-->Arg) of the microsomal epoxide hydrolase gene using fluorescence PCR method combined with melting curves analysis

An association between exon 3 polymorphisms of the gene encoding microsomal epoxide hydrolase (mEH) and susceptibility to the development of chronic obstructive pulmonary disease (COPD) has been described. We have developed two methods for detecting polymorphisms at exons 3 (Tyr113-->His) and 4 (His139-->Arg) of the mEH gene based on different melting temperatures (T(m)) of fluorescent-labeled oligonucleotide hybridization probes using single-step assays that combine fluorescence PCR and melting curve analysis (LightCycler methodology). DNA was extracted from blood in 79 COPD patients and 146 healthy controls. Results were compared with those obtained by restriction fragment length polymorphism (RFLP) analysis to detect Tyr113His variants and a single-strand conformation polymorphism (SSCP) assay for His139Arg detection. The T(m) of the exon 3 polymorphisms were 61.3 degrees C for Tyr113 (wild type) and 67.5 degrees C for His113 (mutant). The T(m) values of the exon 4 polymorphisms were 67.5 degrees C for His139 (wild type) and 59.2 degrees C for Arg139 (mutant). The within- and between-run melting peaks for the same allele differed by less than 0.5 degrees C for both the exon 3 and the exon 4 polymorphisms. Thus, melting analysis allowed easy and unambiguous assignment of genotyping by means of the respective melting curves. The proportion of individuals who were homozygous mutant for exon 3 was significantly higher in the COPD group than in the control group (p=0.004). LightCycler fluorescence genotyping of exon 4 polymorphisms correlated perfectly with SSCP results. RFLP assay classified 2 patients as homozygous mutant while LightCycler analysis genotyped them as heterozygous. DNA analysis by PCR and sequencing confirmed the LightCycler result. These high-speed (about 40 min for 32 samples), highly sensitive, and specific small-volume assays with low labor requirements hold great promise as tools for rapid detection of COPD susceptibility.     

Calendar

Telomeres cap the ends of chromosomes and are essential for the protection of chromosomes, as well as restricting the replicative potential of a cell. These functions are achieved by the regulation of telomeric repeat length, making the measurement of telomere length a useful aid in the elucidation of the replicative history and potential of cells. Previously published techniques employed either hybridization or flow cytometry methods, which are technically demanding and time-consuming. In 2002, R. M. Cawthon published a real-time polymerase chain reaction (PCR)-based method for telomere length measurement using the Applied Biosystems Prism 7700 sequence detection system. The technique measures the factor by which the ratio of telomere repeat copy number to single-gene copy number differs between a sample and that of a reference deoxyribonucleic acid sample. In many laboratories worldwide, including ours, real-time PCR is carried out using the Roche LightCycler, as opposed to the AB Prism 7700 system. This benchmark details the modifications to Cawthon’s method and describes the parameters and reagents required to measure telomere length using the Roche LightCycler.     

Comparative sensitivity of four different cell lines for the isolation of Coxiella burnetii

Coxiella burnetii is an obligate intracellular bacterium that causes the disease Q-fever. This is usually diagnosed by serology (immunofluorescence assay) and/or PCR detection of C.?burnetii DNA. However, neither of these methods can determine the viability of the bacterium. Four different cell lines were compared for their ability to amplify very low numbers of viable C.?burnetii. Two different isolates of C.?burnetii were used. For the Henzerling isolate, DH82 (dog macrophage) cells were the most sensitive with an ID (50) (dose required to infect 50% of cell cultures) of 14.6 bacterial copies. For the Arandale isolate, Vero (monkey epithelial) cells were the most sensitive with an ID (50) of less than one bacterium in a 100-μL inoculum. The Vero cell line appeared highly useful as vacuoles could be seen microscopically in unstained infected cells. The findings of this study favour the use of Vero and DH82 tissue culture cell lines for isolation and growth of C.?burnetii in vitro. The other cell lines, XTC-2 and L929, were less suitable.     

耐除草剂玉米G1105E-823C定性检测方法

转基因耐除草剂玉米G1105E-823C是经过改造的转mG2-aroA基因耐草甘膦玉米新品系,具有更高的草甘膦耐受性,目前已完成生产性试验,具有重要的产业化应用前景。但目前尚无针对G1105E-823C的转化体特异性检测方法的相关报道,这十分不利于对该品系的检测及监管。基于此,以G1105E-823C转化体特异性序列为靶标,建立了转基因耐除草剂玉米G1105E-823C的普通PCR和实时荧光PCR定性检测方法。结果表明,2种方法均能检测出转基因耐除草剂玉米G1105E-823C转化体成分,且具有较高的特异性。普通PCR检测方法检出限达0.1%,实时荧光PCR检测方法检出限达0.05%。研究建立的2种定性检测方法为转基因耐除草剂玉米G1105E-823C的精准检测提供了新的技术手段,可为农业转基因监管提供技术支撑。     

Rapid detection of Yersinia pestis with multiplex real-time PCR assays using fluorescent hybridisation probes

The objective of the present study was to establish a system of real-time polymerase chain reactions (PCRs) for the specific detection of Yersinia pestis using the LightCycler (LC) instrument. Twenty-five strains of Y. pestis, 94 strains of other Yersinia species and 33 clinically relevant bacteria were investigated. Assays for the 16S rRNA gene target and the plasminogen activator gene (resides on the 9.5-kb plasmid) and for the Y. pestis murine toxin gene and the fraction 1 antigen gene (both on the 100-kb plasmid) were combined for the use in two multiplex assays including an internal amplification control detecting bacteriophage lambda-DNA. Applying these multiplex assays, Y. pestis was selectively identified; other bacteria yielded no amplification products. The lower limit of detection was approximately 0.1 genome equivalent. Rat or flea DNA had no inhibitory effects on the detection of Y. pestis. The results obtained using the multiplex real-time assays showed 100% accuracy when compared with combinations of conventional PCR assays. We developed and evaluated a highly specific real-time PCR strategy for the detection of Y. pestis, obtaining results within 3 h including DNA preparation.     

Experimental inoculation of male rats with Coxiella burnetii: successful infection but no transmission to cage mates

Beginning in 2007, the largest human Q fever outbreak ever described occurred in the Netherlands. Dairy goats from intensive farms were identified as the source, amplifying Coxiella burnetii during gestation and shedding large quantities during abortions. It has been postulated that wild rodents are reservoir hosts from which C. burnetii can be transmitted to domestic animals and humans. However, little is known about the infection dynamics of C. burnetii in wild rodents. The aim of this study was to investigate whether brown rats (Rattus norvegicus) can be experimentally infected with C. burnetii and whether transmission to a cage mates occurs. Fourteen male brown rats (wild type) were intratracheally or intranasally inoculated with a Dutch C. burnetii isolate obtained from a goat. At 3 days postinoculation, a contact rat was placed with each inoculated rat. The pairs were monitored using blood samples and rectal and throat swabs for 8 weeks, and after euthanasia the spleens were collected. Rats became infected by both inoculation routes, and detection of C. burnetii DNA in swabs suggests that excretion occurred. However, based on the negative spleens in PCR and the lack of seroconversion, none of the contact animals was considered infected; thus, no transmission was observed. The reproduction ratio R(0) was estimated to be 0 (95% confidence interval = 0 to 0.6), indicating that it is unlikely that rats act as reservoir host of C. burnetii through sustained transmission between male rats. Future research should focus on other transmission routes, such as vertical transmission or bacterial shedding during parturition.     

Efficient method of cloning the obligate intracellular bacterium Coxiella burnetii

Coxiella burnetii is an obligate intracellular bacterium that replicates in a large lysosome-like parasitophorous vacuole (PV). Current methods of cloning C. burnetii are laborious and technically demanding. We have developed an alternative cloning method that involves excision of individual C. burnetii-laden PVs from infected cell monolayers by micromanipulation. To demonstrate the cloning utility and efficiency of this procedure, we coinfected Vero cells with isogenic variants of the Nine Mile strain of C. burnetii. Coinhabited PVs harboring Nine Mile phase II (NMII) and Nine Mile phase I (NMI) or Nine Mile crazy (NMC) were demonstrated by immunofluorescence. PVs were then randomly excised from cells coinfected with NMI and NMC by micromanipulation, and PVs harboring both strains were identified by PCR. Fresh Vero cells were subsequently infected with organisms from coinhabited PVs, and the PV excision and PCR screening process was repeated. Without exception, PVs obtained from second-round excisions contained clonal populations of either NMII or NMC, demonstrating that micromanipulation is an efficient and reproducible procedure for obtaining C. burnetii clones.     

Detection and identification by real time PCR/RFLP analyses of Cryptosporidium species from human faeces

AIMS: To detect a wide range of Cryptosporidium species from human faeces by analysis of the Cryptosporidium oocyst wall protein gene by PCR. METHODS AND RESULTS: The nested-assay comprised an initial amplification using a conventional thermocycler followed by real time PCR using a LightCycler with SYBR Green I for the characterization of the amplicons. The technique uses four sets of primers composed of five to six oligonucleotides with one to six base differences corresponding to the inter-species sequence differences of the gene fragment. Restriction fragment length polymorphism analysis identified Cryptosporidium hominis and C. parvum. The assay was evaluated using DNA extracted from purified material and faecal specimens containing a range of potential pathogens (including Cryptosporidium). The assay was specific, sensitive, reproducible and rapid. CONCLUSIONS: This unique technique enables the rapid detection of a range of polymorphic COWP gene sequences directly from faeces using real time PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates a novel approach to identification of Cryptosporidium species and the identification of C. hominis and C. parvum. The technique may be especially useful for the analysis of environmental samples which are likely to contain heterogeneous mixtures of Cryptosporidium species.     

Quantification and optimization of Candida albicans DNA in blood samples using Real- Time PCR

Background:

Candida albicans (C. albicans) is a major cause of candidaemia in people with impaired immunity. Blood culture is a “gold standard” for candidaemia detection but is time-consuming and relatively insensitive. We established a real-time PCR assay for C. albicans detection in blood by LightCycler PCR and melting curve analysis.

Methods:

Five milliliter blood samples from healthy volunteers were spiked with 10⁰-10⁶ C. albicans cells to determine the detection limit of our method. DNA was extracted from whole blood using glass beads and the QIAamp DNA Blood Mini Kit (Qiagen, Hilden Germany). DNA from C. albicans isolates were amplified with primers and inserted into Escherichia coli (E. coli) DH5α.1 cells with the TA cloning vector (Invitrogen). The plasmid was used for standardization and optimization. A quantitative PCR assay with the LightCycler amplification and detection system based on fluorescence resonance energy transfer (FRET) with two different specific probes was established. To assess the precision and reproducibility of real-time PCR the intra-assay precision was determined in six consecutive assays.

Results:

No cross-reactivity of the hybridization probes with the DNA of non-C. albicans species or human genomic DNA was observed, which confirmed its 100% specificity. The minimum limit detected was one C. albicans cell or 10⁰ CFU/ml (10 fg) per PCR reaction. The real-time PCR efficiency rate for Candida was high (E = 1.95). Melting curve analysis of C. albicans showed a specific melting peak temperature of 65.76 °C.

Conclusion:

The real-time PCR assay we developed is highly specific and sufficiently sensitive to detect the fungal load for early diagnosis of invasive candidiasis. Key Words: Invasive candidiasis, Real-time PCR, Candida albicans     

Real-time PCR test for detection and quantification of human polyomavirus BK (BKV) DNA

The human polyomavirus BK (BKV) is wide-spread pathogen, associated with urogenital tract disorders or even nephropathy in immunosuppressed patients. Nowadays molecular detection by real-time PCR (qPCR) is recognized as a method-of-choice for detecting human polyomaviruses in clinical samples. The aim of the study was development of real-time PCR assay for detection and quantification of polyomavirus BK DNA in clinical samples, using specific primers targeting a viral DNA VP3 gene and a TaqMan hydrolyzing probe. The analytical sensitivity of assay was tested using serial dilutions of BKV DNA in range between 13500 and 15 copies/ml. 27 urine samples and 23 plasma samples taken from a group of 22 adult recipients of allogeneic HSCT were tested for the presence of polyomavirus BK in the LightCycler system. Described in-house real-time PCR assay detected BKV DNA in 8 specimens (6 urine and 2 plasma). Detected average viral load was 170 copies/ml for plasma and 1250 copies/ml for urine samples, respectively. The results of this study show that developed TaqMan-based probe qPCR assay is very reliable and valuable for detection and quantification of BKV DNA, both in urine and plasma samples. These data, combined with its rapid turnaround time for results and decreased hands-on time, make the LightCycler PCR assay highly suitable for the rapid diagnostics of polyomavirus BK infections in the clinical laboratory.