A real-time PCR assay for detection and quantification of Mycoplasma agalactiae DNA

AIMS: The aim of this study was to develop a rapid, sensitive, specific tool for detection and quantification of Mycoplasma agalactiae DNA in sheep milk samples. METHODS AND RESULTS: A real-time polymerase chain reaction (PCR) assay targeting the membrane-protein 81 gene of M. agalactiae was developed. The assay specifically detected M. agalactiae DNA without cross-amplification of other mycoplasmas and common pathogens of small ruminants. The method was reproducible and highly sensitive, providing precise quantification of M. agalactiae DNA over a range of nine orders of magnitude. Compared with an established PCR assay, the real-time PCR was one-log more sensitive, detecting as few as 10(1) DNA copies per 10 microl of plasmid template and 6.5x10(0) colour changing units of reference strain Ba/2. CONCLUSIONS: The real-time PCR assay is a reliable method for the detection and quantification of M. agalactiae DNA in sheep milk samples. The assay is more sensitive than gel-based PCR protocols and provides quantification of the M. agalactiae DNA contained in milk samples. The assay is also quicker than traditional culture methods (2-3 h compared with at least 1 week). SIGNIFICANCE AND IMPACT OF THE STUDY: The established real-time PCR assay will help study the patterns of shedding of M. agalactiae in milk, aiding pathogenesis and vaccine efficacy studies.     

Development and evaluation of a real-time quantitative PCR assay for Aspergillus flavus

Aspergillus flavus is a ubiquitous mold and the most common mold contaminating foodstuffs. Many strains of A. flavus produce aflatoxins. In addition it is an allergen and an opportunistic pathogen of animals and plants. A. flavus often is underestimated in traditional culture analyses due to the expertise required and the cost associated with speciating members of the genus Aspergillus. The goal of this study was to develop and validate a primer and probe set for the rapid detection and quantitation of A. flavus in pure culture using real-time quantitative polymerase chain reaction (QPCR) amplification. Unique DNA regions were located in the genome of the target organism by sequence comparison with the GenBank database, and several candidate oligonucleotides were identified from the scientific literature for potential use with the TaqMan QPCR technology. Three primer and probe sets were designed and validated for specificity and sensitivity in laboratory experiments. Initial screening to test for sensitivity was performed with seven A. flavus isolates and selected nontarget fungi. Specificity testing was conducted with the selected primer and probe set, which amplified all nine A. flavus isolates tested, including an aflatoxin producing strain. The primers did not amplify DNA extracted from 39 other fungal species (comprising 16 genera), including 18 other Aspergillus species and six Penicillium species. No amplification of human or bacterial DNA was observed; however cross-reactivity was observed with Aspergillus oryzae. PCR analysis of DNA dilutions and the use of an internal positive control demonstrated that 67% of the fungal DNA samples assayed contained PCR inhibitors. The assay validated for the target organism is capable of producing PCR results in less than 1 h after DNA extraction. The results of this research demonstrate the capabilities of QPCR for the enhanced detection and enumeration of fungi of significance to human health.     

Molecular-beacon multiplex real-time PCR assay for detection of Vibrio cholerae

A multiplex real-time PCR assay was developed using molecular beacons for the detection of Vibrio cholerae by targeting four important virulence and regulatory genes. The specificity and sensitivity of this assay, when tested with pure culture and spiked environmental water samples, were high, surpassing those of currently published PCR assays for the detection of this organism.     

A multiplex real-time PCR assay for detection of oseltamivir-resistant strains of influenza virus

Influenza is a contagious disease of humans and animals caused by viruses belonging to the Orthomyxoviridae family. The influenza A virus genome consists of negative sense, single-stranded, segmented RNA. Influenza viruses are classified into subtypes based on two surface antigens known as hemagglutinin (H) and neuraminidase (N). The main problem with influenza A viruses infecting humans is drug resistance, which is caused by antigenic changes. A few antiviral drugs are available, but the most popular is the neuraminidase inhibitor — oseltamivir. The resistance against this drug has probably developed through antigenic drift by a point mutation in one amino acid at position 275 (H275Y). In order to prevent a possible influenza pandemic it is necessary to develop fast diagnostic tests. The aim of this project was to develop a new test for detection of influenza A virus and determination of oseltamivir resistance/sensitivity in humans. Detection and differentiation of oseltamivir resistance/sensitivity of influenza A virus was based on real-time PCR. This test contains two TaqMan probes, which work at different wavelengths. Application of techniques like multiplex real-time PCR has greatly enhanced the capability for surveillance and characterization of influenza viruses. After its potential validation, this test can be used for diagnosis before treatment.     

Nested real-time PCR for hepatitis A detection

Aims:  The hepatitis A virus (HAV) is one of the most important human foodborne pathogens causing a number of worldwide outbreaks each year. The detection of HAV in food samples remains a complex issue, because commonly used detection tools, such as conventional or even real-time PCR assays, are often unable to detect HAV with sufficient sensitivity. The aims of this study were to develop highly sensitive and specific nested real-time PCR (NRT-PCR)-based method for HAV detection in food and to compare it with currently available methods.
Methods and Results:  By combining conventional PCR, nested PCR and real-time PCR techniques, we have developed a specific NRT-PCR assay for the detection of HAV. The procedure involves two consecutive PCRs, the first of which is performed as a conventional RT-PCR using primers specific for HAV 5' noncoding region. The second reaction involves a real-time PCR using a nested primer pair specific for the first PCR product and a TaqMan probe.
Conclusions:  We have developed a novel NRT-PCR method capable of detecting as little as 0·2 PFU of HAV, which is significantly more sensitive than any other PCR technique tested in our system.
Significance and Impact of the Study:  NRT-PCR provides a potentially useful method for detecting HAV at extremely low levels, as frequently found in food samples, and can be potentially adopted as a regulatory method to ensure food safety.     

A real-time PCR assay for detection of isoniazid resistance in Mycobacterium tuberculosis clinical isolates

A real-time PCR genotypic assay was developed for the detection of isoniazid (INH) resistance in Mycobacterium tuberculosis. The assay detects mutations C(-15)T and, possibly, G(-24)T in the regulatory region of the inhA gene and proved as sensitive and specific as nucleotide sequencing in all the clinical isolates tested. Our assays mapped the mutations efficiently in 10 out of 35 resistant isolates, thereby covering 29% of all resistant strains.     

A real-time PCR assay for DNA-methylation using methylation-specific blockers

DNA methylation-based biomarkers have been discovered that could potentially be used for the diagnosis of cancer by detection of circulating, tumor-derived DNA in bodily fluids. Any methylation detection assay that would be applied to these samples must be capable of detecting small amounts of tumor DNA in the presence of background normal DNA. We have developed a real-time PCR assay, called HeavyMethyl, that is well suited for this application. HeavyMethyl uses methylation-specific oligonucleotide blockers and a methylation-specific probe to achieve methylation-specific amplification and detection. We tested the assays on unmethylated and artificially methylated DNA in order to determine the limit of detection. After careful optimization, our glutathione-S-transferase pi1 and Calcitonin assays can amplify as little as 30 and 60 pg of methylated DNA, respectively, and neither assay amplifies unmethylated DNA. The Calcitonin assay showed a highly significant methylation difference between normal colon and colon adenocarcinomas, and methylation was also detected in serum DNA from colon cancer patients. These assays show that HeavyMethyl technology can be successfully employed for the analysis of very low concentrations of methylated DNA, e.g. in serum of patients with tumors.     

Rapid detection of Enterobacter sakazakii using TaqMan real-time PCR assay

Enterobacter sakazakii is an emerging food pathogen, which induces severe meningitis and sepsis in neonates and infants, with a high fatality rate. The disease is generally associated with the ingestion of contaminated infant formula. In this study, we describe the development of a real-time PCR protocol to identify E. sakazakii using a TaqMan probe, predicated on the nucleotide sequence data of the 16S rRNA gene obtained from a variety of pathogens. To detect E. sakazakii, four primer sets and one probe were designed. Five strains of E. sakazakii and 28 non-E. sakazakii bacterial strains were used in order to ensure the accuracy of detection. The PCR protocol successfully identified all of the E. sakazakii strains, whereas the 28 non-E. sakazakii strains were not detected by this method. The detection limits of this method for E. sakazakii cells and purified genomic DNA were 2.3 CFU/assay and 100 fg/assay, respectively. These findings suggest that our newly developed TaqMan real-time PCR method should prove to be a rapid, sensitive, and quantitative method for the detection of E. sakazakii.     

A novel real-time PCR assay for the detection of Helicobacter pullorum-like organisms in chicken products

A novel real-time PCR assay was developed for the direct detection in food of Helicobacter pullorum-like bacteria, which are occasionally associated with human enteric disease. Experiments using control strains showed that the realtime PCR assay was specific and reproducible, with a detection level of 1 colony-forming unit (CFU)/g. The assay was then applied to determine contamination rates in 30 samples of three types of chicken-meat products obtained from five retail outlets in Spain (Valencia); all of the samples were initially considered to be culture-negative for Helicobacter even after an enrichment period. H.pullorum-like DNA was detected in seven out of ten chicken carcasses and in one chicken-burger sample (without enrichment), as well as in one liver sample (after enrichment). Sequencing of three randomly selected PCR products confirmed concordance (99% homology) with the H. pullorum 16S rDNA gene. The advantages of real-time PCR over conventional PCR assays are the improved detection level, speed of testing, and validation of specificity by melting-point analysis. The fact that bacteria are frequently present in chicken carcasses sold in retail stores highlights the importance of more widely monitoring contamination rates. The novel assay described herein allows better assessment of potential human health risks posed by H. pullorum.     

A real-time PCR assay for the detection of Campylobacter jejuni in foods after enrichment culture

A real-time PCR assay was developed for the quantitative detection of Campylobacter jejuni in foods after enrichment culture. The specificity of the assay for C. jejuni was demonstrated with a diverse range of Campylobacter species, related organisms, and unrelated genera. The assay had a linear range of quantification over six orders of magnitude, and the limit of detection was approximately 12 genome equivalents. The assay was used to detect C. jejuni in both naturally and artificially contaminated food samples. Ninety-seven foods, including raw poultry meat, offal, raw shellfish, and milk samples, were enriched in blood-free Campylobacter enrichment broth at 37 degrees C for 24 h, followed by 42 degrees C for 24 h. Enrichment cultures were subcultured to Campylobacter charcoal-cefoperazone-deoxycholate blood-free selective agar, and presumptive Campylobacter isolates were identified with phenotypic methods. DNA was extracted from enrichment cultures with a rapid lysis method and used as the template in the real-time PCR assay. A total of 66 samples were positive for C. jejuni by either method, with 57 samples positive for C. jejuni by subculture to selective agar medium and 63 samples positive in the real-time PCR assay. The results of both methods were concordant for 84 of the samples. The total time taken for detection from enrichment broth samples was approximately 3 h for the real-time PCR assay, with the results being available immediately at the end of PCR cycling, compared to 48 h for subculture to selective agar. This assay significantly reduces the total time taken for the detection of C. jejuni in foods and is an important model for other food-borne pathogens.     

WSSV和IHHNV二重实时荧光PCR检测方法的建立

根据基因库中对虾白斑综合征病毒WSSV(AF369029)和传染性皮下及造血器官坏死病毒IHHNV(AF218226)基因序列,设计了WSSV和IHHNV的两对特异性引物和两条用不同荧光基团标记的TaqMan探针。对反应条件和试剂浓度进行优化,建立了能够同时检测WSSV和IHHNV的二重实时荧光PCR方法。该方法特异性好,对WSSV和IHHNV的检测敏感性分别达到2和20个模板拷贝数;此外抗干扰能力强,对WSSV和IHHNV不同模板浓度进行组合,仍可有效地同时检测这二个病毒。对保存的30份经常规PCR检测仅为WSSV或IHHNV阳性的样品进行二重实时荧光PCR检测,结果都为阳性,其中1份为WSSV和IHHNV混合感染。本研究建立的二重实时荧光PCR方法用于WSSV和IHHNV的检测具有特异、敏感、快速、定量等优点。     

Multiplex real-time PCR assay for simultaneous detection of Omphalotus guepiniformis and Lentinula edodes

A rapid multiplex real-time PCR assay was developed to achieve highly specific, simultaneous detection of two kinds of mushrooms, Omphalotus guepiniformis and Lentinula edodes. Primers and TaqMan minor groove binder probes were designed according to the internal transcribed spacers 1-5.8S region of rDNA and evaluated by the specificity for fruiting bodies of 17 O. guepiniformis, 16 L. edodes and samples from 57 other species. DNA extracts of all the target species had positive signals with no cross-reaction, the limit of detection being 0.00025 ng of DNA. Threshold cycle (Ct) values for raw and processed fruiting bodies and for fruiting bodies (1% (w/w)) mixed with foodstuffs or artificial gastric juice contents ranged from 17.16 to 26.60 for both examined species. This new assay proved specific to the target species, highly sensitive, and applicable to processed food samples and gastric juice contents, making it useful for rapidly identifying O. guepiniformis and L. edodes.     

A duplex real-time PCR assay for detection of drug-induced mitochondrial DNA depletion in HepG2 cells

Screening of drug-induced mitochondrial DNA (mtDNA) depletion during early preclinical drug development is of major interest. Here we describe the establishment of a novel duplex calibrator-normalized real-time polymerase chain reaction (PCR) assay for rapid and reliable quantification of mtDNA in HepG2 cells. This assay involves quantification of an mtDNA target gene (cytochrome b) relative to a nuclear DNA (nDNA) reference gene (β-actin) in one tube. The assay was evaluated for its precision, linearity, and reproducibility, and reliable detection of mtDNA depletion was demonstrated. Using this novel real-time PCR assay, drug-induced mtDNA depletion could be accurately detected.     

Development of real-time PCR assay for differential detection and quantification for multiple Babesia microti-genotypes

We have developed a real-time PCR assay that can rapidly and differentially detect and quantify four genotypes of small subunit ribosomal RNA gene (SSUrDNA) of Babesia microti (Kobe-, Otsu-, Nagano- and US-types). In this assay, four genotype-specific pairs of primers targeted on internal transcribed spacer (ITS) 1 or 2 sequences were used and amplicons by each pair of primers were quantitatively detected by fluorescent SYBR Green I. The four genotype-specific pairs of primers displayed the high specificity for homologous genotype DNA. The standard curves of cycle threshold (Ct) values versus amount of target DNA per reaction (log) for all four genotypes were linear and the correlation coefficient (Rsq) values for the curves were from 0.970 to 0.997. The standard curves were almost identical even in the presence of heterologous genotype DNA. This assay could detect 10-30 fg purified DNA (equivalent to the amount of 1-5 parasite DNA) of each genotype B. microti. This assay could also detect each genotype B. microti infection in blood with 3×10(-6)%-1×10(-5)% parasitemia. This assay was applicable to field rodent and tick samples to reveal mixed infection in several samples, for which a single genotype of B. microti had been detected by direct sequencing analyses in our previous studies. This assay also seemed to be applicable to clinical human samples, showing Kobe-type positive results for the first Japanese babesiosis patient and the asymptomatic donor, both infected with Kobe-type B. microti.     

A new approach to apple proliferation detection: a highly sensitive real-time PCR assay

The present paper describes a new approach for diagnosis of apple proliferation (AP) phytoplasma in plant material using a multiplex real-time PCR assay simultaneously amplifying a fragment of the pathogen 16S rRNA gene and the host, Malus domestica, chloroplast gene coding for tRNA leucine. For the first time, such an approach, with an internal analytical control, is described in a diagnostic procedure for plant pathogenic phytoplasmas enabling distinction between uninfected plant material and false-negative results caused by PCR inhibition. Pathogen detection is based on the highly conserved 16S rRNA gene to ensure amplification of different AP phytoplasma strains. The newly designed primer/probe set allows specific detection of all examined AP strains, without amplifying other fruit tree phytoplasmas or more distantly related phytoplasma strains. Apart from its specificity, real-time PCR with serial dilutions of initial template DNA ranging over almost five orders of magnitude (undiluted to 80,000-fold diluted) demonstrated linear amplification over the whole range, while conventional PCR showed a reliable detection only up to 500-fold or 10,000-fold dilutions, respectively. Compared to existing analytical diagnostic procedures for phytoplasmas, a rapid, highly specific and highly sensitive diagnostic method becomes now available.     

A TaqMan-based real-time PCR assay for the specific detection and quantification of Leuconostoc mesenteroides in meat products

A new real-time PCR procedure was developed for the specific detection and quantification of Leuconostoc mesenteroides in meat products. It is a TaqMan assay based on 23S rRNA gene targeted primers and probe. Specificity was evaluated using purified DNA from 132 strains: 102 lactic acid bacteria (LAB), including 57 reference strains and 46 food isolates, belonging to genus Leuconostoc and related genera, and 30 non-LAB strains. Quantification was linear over at least 5 log units using both serial dilutions of purified DNA and calibrated cell suspensions from Leuconostoc mesenteroides ssp. dextranicum CECT 912T. This assay was able to detect at least five genomic equivalents, using purified DNA or 59 CFU per reaction when using calibrated cell suspensions. It performed successfully when tested on an artificially inoculated meat product, with a minimum threshold of 10(4) CFU g(-1) for the accurate quantification of Leuconostoc mesenteroides.     

A nested real-time PCR assay has an increased sensitivity suitable for detection of viruses in aerosol studies

Aims:  Influenza is commonly spread by infectious aerosols; however, detection of viruses in aerosols is not sensitive enough to confirm the characteristics of virus aerosols. The aim of this study was to develop an assay for respiratory viruses sufficiently sensitive to be used in epidemiological studies.
Method:  A two-step, nested real-time PCR assay was developed for MS2 bacteriophage, and for influenza A and B, parainfluenza 1 and human respiratory syncytial virus. Outer primer pairs were designed to nest each existing real-time PCR assay. The sensitivities of the nested real-time PCR assays were compared to those of existing real-time PCR assays. Both assays were applied in an aerosol study to compare their detection limits in air samples.
Conclusions:  The nested real-time PCR assays were found to be several logs more sensitive than the real-time PCR assays, with lower levels of virus detected at lower Ct values. The nested real-time PCR assay successfully detected MS2 in air samples, whereas the real-time assay did not.
Significance and Impact of the Study:  The sensitive assays for respiratory viruses will permit further research using air samples from naturally generated virus aerosols. This will inform current knowledge regarding the risks associated with the spread of viruses through aerosol transmission.     

产赭曲霉毒素A黑曲霉的PCR法检测

【目的】快速检测产赭曲霉毒素A(OTA)的黑曲霉。【方法】根据黑曲霉(Aspergillus niger)CBS513.88中An15g07920基因编码聚酮合酶的酰基转移酶(AT)域设计引物,建立针对产OTA黑曲霉的聚合酶链式反应(PCR)检测方法。【结果】对72株曲霉属菌株(黑曲霉、炭黑曲霉、赭曲霉、佩特曲霉、寄生曲霉和塔宾曲霉)进行检测,发现产OTA的黑曲霉能够扩增出特异性条带,而产OTA的其它菌株不能扩增出条带;检测出3株假阳性的产OTA黑曲霉,实时定量PCR分析此3株菌中An15g07920的同源基因表达情况,发现在产毒条件下可正常表达,排除了因基因无法表达导致假阳性的可能。本方法的检测灵敏度为25 pg的DNA含量,在污染所试农产品孢子浓度大于4.0×10~4–4.0×10~5个/g时可有效检测出产毒菌株。【结论】本方法虽会产生4%的假阳性结果,但是仍可作为产毒黑曲霉有效的快速检测方法,并在农产品污染产毒黑曲霉时进行有效预警。     

Adapting a conventional PCR assay for Toxoplasma gondii detection to real-time quantitative PCR including a competitive internal control

We have developed a quantitative PCR assay (LightCycler* using the pair of primers JW58 and JW59 for the detection of the 35-fold repeated B gene of oxoplasma gondii. This real-time PCR, using fluorescence resonance energy transfert (FRET) hybridization probes, allows the quantification of . gondii with several technical requirements not previously described: i) an internal amplification control (co-amplified in a single tube with the same primers), ii) Uracil-N-Glycosylase and iii) a standard curve corresponding to a serial dilution from a calibrated suspension of T. gondii ranging from 40 to 4.106( )parasites in one ml of amniotic fluid (1 to 105( ) . gondii/PCR). In artificial samples, one parasite could be detected if at least three reactions were performed.