Comparison of an immunochromatographic method and the TaqMan E. coli O157:H7 assay for detection of Escherichia coli O157:H7 in alfalfa sprout spent irrigation water and in sprouts after blanching

An immunochromatographic-based assay (Quixtrade mark E. coli O157 Sprout Assay) and a polymerase chain reaction (PCR)-based assay (TaqMan E. coli O157:H7 Kit) were used to detect Escherichia coli O157:H7 strain 380-94 in spent irrigation water from alfalfa sprouts grown from artificially contaminated seeds. Ten, 25, 60, or 100 seeds contaminated by immersion for 15 min in a suspension of E. coli O157:H7 at concentrations of 10(6) or 10(8) cfu/ml were mixed with 20 g of non-inoculated seeds in plastic trays for sprouting. The seeds were sprayed with tap water for 15 s every hour and spent irrigation water was collected at intervals and tested. E. coli O157:H7 was detected in non-enriched water by both the TaqMan PCR (30 of 30 samples) and the immunoassay (9 of 24 samples) in water collected 30 h from the start of the sprouting process. However, enrichment of the spent irrigation water in brain heart infusion (BHI) broth at 37 degrees C for 20 h permitted detection of E. coli O157:H7 in water collected 8 h from the start of sprouting using both methods, even in trays containing as few as 10 inoculated seeds. The TaqMan PCR assay was more sensitive (more positive samples were observed earlier in the sprouting process) than the immunoassay; however, the immunoassay was easier to perform and was more rapid. At 72 h after the start of the sprouting process, the sprouts were heated at 100 degrees C for 30 s to determine the effectiveness of blanching for inactivation of E. coli O157:H7. All of the 32 samples tested with the TaqMan assay and 16 of 32 samples tested with the Quixtrade mark assay gave positive results for E. coli O157:H7 after enrichment of the blanched sprouts at 37 degrees C for 24 h. In addition, the organism was detected on Rainbow Agar O157 in 9 of 32 samples after 24 h of enrichment of the blanched sprouts. In conclusion, E. coli O157:H7 was detected in spent irrigation water collected from sprouts grown from artificially contaminated seeds by both the TaqMan and Quixtrade mark assays. The data also revealed that blanching may not be effective to completely inactivate all the E. coli O157:H7 that may be present in sprouts.     

Molecular and bioassay-based detection of Toxoplasma gondii oocyst uptake by mussels (Mytilus galloprovincialis)

Toxoplasma gondii is associated with morbidity and mortality in a variety of marine mammals, including fatal meningoencephalitis in the southern sea otter (Enhydra lutris nereis). The source(s) of T. gondii infection and routes of transmission in the marine environment are unknown. We hypothesise that filter-feeding marine bivalve shellfish serve as paratenic hosts by assimilation and concentration of infective T. gondii oocysts and their subsequent predation by southern sea otters is a source of infection for these animals. We developed a TaqMan PCR assay for detection of T. gondii ssrRNA and evaluated its usefulness for the detection of T. gondii in experimentally exposed mussels (Mytilus galloprovincialis) under laboratory conditions. Toxoplasma gondii-specific ssrRNA was detected in mussels as long as 21 days post-exposure to T. gondii oocysts. Parasite ssrRNA was most often detected in digestive gland homogenate (31 of 35, i.e. 89%) compared with haemolymph or gill homogenates. Parasite infectivity was confirmed using a mouse bioassay. Infections were detected in mice inoculated with any one of the mussel sample preparations (haemolymph, gill, or digestive gland), but only digestive gland samples remained bioassay-positive for at least 3 days post-exposure. For each time point, the total proportion of mice inoculated with each of the different tissues from T. gondii-exposed mussels was similar to the proportion of exposed mussels from the same treatment groups that were positive via TaqMan PCR. The TaqMan PCR assay described here is now being tested in field sampling of free-living invertebrate prey species from high-risk coastal locations where T. gondii infections are prevalent in southern sea otters.     

A real-time PCR for SARS-coronavirus incorporating target gene pre-amplification

An enhanced polymerase chain reaction (PCR) assay to detect the coronavirus associated with severe acute respiratory syndrome (SARS-CoV) was developed in which a target gene pre-amplification step preceded TaqMan real-time fluorescent PCR. Clinical samples were collected from 120 patients diagnosed as suspected or probable SARS cases and analyzed by conventional PCR followed by agarose gel electrophoresis, conventional TaqMan real-time PCR, and our enhanced TaqMan real-time PCR assays. An amplicon of the size expected from SARS-CoV was obtained from 28/120 samples using the enhanced real-time PCR method. Conventional PCR and real-time PCR alone identified fewer SARS-CoV positive cases. Results were confirmed by viral culture in 3/28 cases. The limit of detection of the enhanced real-time PCR method was 10(2)-fold higher than the standard real-time PCR assay and 10(7)-fold higher than conventional PCR methods. The increased sensitivity of the assay may help control the spread of the disease during future SARS outbreaks.     

游离线粒体拷贝数检测方法建立及应用

摘要 目的：线粒体在生理和病理过程中都起着重要作用,线粒体破碎后形成的游离线粒体与一系列疾病密切相关。然而,人体内游离线粒体的含量较低很难被稳定抽提且易降解等因素导致游离线粒体拷贝数检测具有极大挑战。本研究拟建立一种快速、准确检测外周血游离线粒体拷贝数定量PCR技术。方法：通过多重荧光定量PCR技术在SLAN?-96S全自动医用PCR分析系统上检测人外周血游离线粒体拷贝数,构建新的游离线粒体检测方案。游离核基因在人体外周血中的稳定性远大于游离线粒体,因此使用多拷贝参考基因YH-1（300拷贝）检测游离核基因作为对照组。结果：成功建立了核基因标准曲线和线粒体标准曲线,并筛选游离线粒体拷贝数检测最佳引物扩增片段长度为82bp、血清有效分离时间在2h内、血清最佳分离方案为1600 r/min 离心10 min再16000 r/min 离心10 min、磁珠法游离核酸抽提试剂盒抽提游离核酸得率最高的新流程。利用新方案对100 例不同年龄段的随机人群外周血抽提游离线粒体拷贝数进行检测,结果显示30-79岁游离线粒体拷贝数与年龄之间的相关性参数为｜R｜= 0.18、P value = 0.077,游离线粒体拷贝数与性别之间的相关性参数为｜R｜= 0.27、P value = 0.061即游离线粒体拷贝数与年龄和性别均无显著相关性,研究结果与报道一致。结论：表明优化后的方案可稳定检测游离线粒体拷贝数,提供了一种快速、准确检测游离线粒体拷贝数的方法。     

Real‐time PCR quantification of Bemisia tabaci (Homoptera: Aleyrodidae) B‐biotype remains in predator guts

The cotton whitefly, Bemisia tabaci (Gennadius) B‐biotype, is fed on by a wide variety of generalist predators, but there is little information on these predator–prey interactions, especially under field conditions. In this study, a real‐time polymerase chain reaction (PCR) assay was developed to quantify B. tabaci B‐biotype remains in predator gut. The B. tabaci B‐biotype genomic DNA copy number was referred to the actual amount of BT1 isolate, the B. tabaci B‐biotype specific DNA fragment. The numbers of BT1 isolate in one B. tabaci B‐biotype egg, individual adult and a single red‐eyed nymph were 2.56 × 10³, 2.56 × 10⁴, and 1.29 × 10⁴ copies, respectively. When Propylaea japonica adults fed on one, two, four, eight or 16 red‐eyed nymphs, the detected numbers of BT1 isolate ranged from 2.77 × 10⁴ to 4.05 × 10⁵ copies, forming a strong linear relationship (R² = 0.9899). Following the consumption of two red‐eyed nymphs, prey DNA was detectable in 100% of P. japonica at t = 0, decreasing to 80.0% and 60.0% after 1–4 h and 8 h of digestion, respectively, with 3.36 × 10⁴–1.25 × 10³ BT1 isolate copies. The predation by field‐collected predators, 26 larvae of P. japonica, and of Harmonia axyridis each, Chrysopa spp. larvae (Chrysopa pallens and C. formosa, 18 individuals in total), and a single adult of Scymnus hoffmanni, 19 adults of Orius sauteri and nine adult spiders (Erigonnidium graminicolum and Neoscona doenitzi), on B. tabaci B‐biotype were quantified. Of the 99 analysed predator individuals, 3.65 × 10²–4.60 × 10⁵ copies of BT1 isolate, equivalent to 0.8–18.8 red‐eyed nymphs were detected. These results suggest that TaqMan real‐time PCR technology may provide a rapid and sensitive method for quantifying B. tabaci B‐biotype remains in predator guts and will be invaluable in assessing the food web relationship between prey and arthropod predators.     

实时荧光定量检测中国实验用小型猪肝脏CYP3A29 mRNA表达水平

CYP3A29是猪肝脏最重要的药物代谢关键酶。研究中国实验用小型猪肝脏CYP3A29 mRNA的表达特性对于评估其是否适宜于作为人CYP3A4介导的药理学研究动物模型具有一定意义。以b-actin作校正, 利用TaqMan定量技术对巴马香猪、贵州小型香猪肝脏CYP3A29 mRNA表达水平进行检测, 并以荣昌猪作为对照。结果表明, 巴马香猪、贵州小型香猪、荣昌猪肝脏CYP3A29 mRNA表达水平与报道的人肝脏CYP3A4相近; 三品系(种)猪间肝脏CYP3A29 mRNA表达水平较为接近, 但品系(种)内个体间变异较大。提示巴马香猪、贵州小型香猪作为药物评价的实验动物具有一定可行性。     

Use of TaqMan gene probe for real-time monitoring of acidophilic hydrogen-producing bacteria

Using a TaqMan gene probe targeting the 16S rDNA of acidophilic hydrogen-producing bacteria (HPB), the abundance of this HPB in the biomass was found to increase from 0.02 to 72% in a single batch treating rice slurry waste at pH 4.5 over 130 h. The corresponding abundances were 4.4% in the batch operated at pH 5.0 and 0.01–0.02% at pH 5.5–6.5. During the growth phase, the generation time for the acidophilic HPB at pH 4.5 averaged 3.5 h.     

Optimization of melting analysis with TaqMan probes for detection of KRAS,NRAS, and BRAF mutations

The TaqMan probes that have been long and effectively used in real-time polymerase chain reaction (PCR) may also be used in DNA melting analysis. We studied some factors affecting efficiency of the approach such as (i) number of asymmetric PCR cycles preceding DNA melting analysis, (ii) choice of fluorophores for the multiplex DNA melting analysis, and (iii) choice of sense or antisense TaqMan probes for optimal resolution of wild-type and mutant alleles. We also determined ΔT_m (i.e., the temperature shift of a heteroduplex relative to the corresponding homoduplex) as a means of preliminary identification of mutation type. In experiments with serial dilution of mutant KRAS DNA with wild-type DNA, the limit of detection of mutant alleles was 1.5–3.0%. Using DNA from both tumor and formalin-fixed paraffin-embedded tissues, we demonstrated a high efficiency of TaqMan probes in mono- and multiplex mutation scanning of KRAS, NRAS (codons 12, 13, and 61), and BRAF (codon 600) genes. This cost-effective method, which can be applied to practically any mutation hot spot in the human genome, combines simplicity, ease of execution, and high sensitivity—all of the qualities required for clinical genotyping.     

Validation of real-time RT-PCR assays for mRNA quantification in baboons

Real-time RT-PCR has been used widely, both in fundamental research and in clinical diagnostics, for instance for quantification of RNA levels in human tissues and tissue biopsies. In the present study we provide a strategy to validate primers/probes for real-time RT-PCR quantification of baboon samples. The method is based on the TaqMan system and uses primers/probes that have been designed and validated for human real-time RT-PCR. A prerequisite for the accuracy of this strategy is a similar amplification efficiency between human and baboon PCR reactions. We propose two different methods, i.e. by calculating PCR efficiencies from the slope of a dilution curve or by using the linear regression method, to compare the amplification efficiency between human and baboon samples. In conclusion, by performing a simple validation experiment, real-time PCR assays based on human sequences, which are easily available, can be applied for analysis of baboon samples.