Differentiation of medicinal Codonopsis species from adulterants by polymerase chain reaction-restriction fragment length polymorphism

DNA sequence analysis of rDNA internal transcribed spacer (ITS) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were exploited for their applications in differentiating medicinal species Codonopsis pilosula, C. tangshen, C. modesta, and C. nervosa var. macrantha, from two related adulterants Campanumoea javania and Platycodon grandiflorus. The data demonstrated that the rDNA ITSI and ITSII sequences of the four Codonopsis are highly homologous but not identical, and are significantly different from those of the two adulterants. The sequence difference allows effective and reliable differentiation of Codonopsis from the adulterants by PCR-RFLP.     

Detection of viable Salmonella in ice cream by TaqMan real-time polymerase chain reaction assay combining propidium monoazide

Real-time polymerase chain reaction (PCR) allows rapid detection of Salmonella in frozen dairy products, but it might cause a false positive detection result because it might amplify DNA from dead target cells as well. In this study, Salmonella-free frozen ice cream was initially inoculated with heat-killed Salmonella Typhimurium cells and stored at −18°C. Bacterial DNA extracted from the sample was amplified using TaqMan probe-based real-time PCR targeting the invA gene. Our results indicated that DNA from the dead cells remained stable in frozen ice cream for at least 20 days, and could produce fluorescence signal for real-time PCR as well. To overcome this limitation, propidium monoazide (PMA) was combined with real-time PCR. PMA treatment can effectively prevent PCR amplification from heat-killed Salmonella cells in frozen ice cream. The PMA real-time PCR assay can selectively detect viable Salmonella at as low as 10³ CFU/mL. Combining 18 hours of pre-enrichment with the assay allows for the detection of viable Salmonella at 10⁰ CFU/mL and avoiding the false-positive result of dead cells. The PMA real-time PCR assay provides an alternative specifically for detection of viable Salmonella in ice cream. However, when the PMA real-time PCR assay was evaluated in ice cream subjected to frozen storage, it obviously underestimated the contamination situation of viable Salmonella, which might lead to a false negative result. According to this result, the use of enrichment prior to PMA real-time PCR analysis remains as the more appropriate approach.     

Primary clarithromycin resistance in Italy assessed on Helicobacter pylori DNA sequences by TaqMan real-time polymerase chain reaction

BACKGROUND: Helicobacter pylori clarithromycin resistance is increasing worldwide and different mutations are involved in its mechanisms. Recently, molecular methods have been proposed to assess these mutations. AIM: To assess prevalence of primary clarithromycin resistance in two Italian areas, and the distribution of involved mutations, by using a novel method for real-time polymerase chain reaction. METHODS: Two hundred and thirty-two H. pylori-positive patients undergoing oesophagogastroduodenoscopy in two Italian towns (Rome, centre Italy; Foggia, south Italy) were enrolled. Helicobacter pylori infection was detected by histology, rapid urease and urea breath tests. Clarithromycin resistance was assessed by TaqMan real-time polymerase chain reaction on paraffin-embedded antral biopsies. Results Primary clarithromycin resistance was detected in 62 (26.7%) patients. Its prevalence did not differ between the two areas (31.5%, centre vs. 23.3%, south; P=0.17) and between non-ulcer dyspepsia and peptic ulcer patients (28.4% vs. 20.7%, P=0.2). The A2143G point mutation was detected in 35 (56.4%) patients, A2142G in 14 (22.6%), A2142C in eight (12.9%), whilst a double mutation (A2143G plus A2142C or A2142G) was present in the remaining five (8.1%) cases. CONCLUSIONS: Our study found that primary clarithromycin resistance is highly prevalent in both central and southern Italy, and that A2143G is the most frequent point mutation involved in these areas.     

Molecular authentication of the traditional Chinese medicinal plant Euphorbia pekinensis

The ITS regions of Euphorbia pekinensis and six other Euphorbia species used as adulterants of E. pekinensis were sequenced to differentiate them. The sequences are identical among the individuals in the seven species studies. Diversity in DNA sequences among various species was found ranging from 8.3% to 43.8% in ITS1 and 7.6% to 36.6% in ITS2 region. Furthermore, based on the divergent ITS regions, species-specific primers, JDJp 1 and JDJp 2, were designed in the polymorphic regions of E. pekinensis to distinguish it from adulterants. These ITS-derived primers amplified a 281-bp-specific DNA fragment from E. pekinensis. No amplified product was observed using DNA of six adulterants.     

实时荧光定量PCR在肺结核诊断中的临床应用

目的探讨实时荧光定量聚合酶链反应法(FQ-PCR荧光定量)对肺结核诊断的临床应用价值。方法用直接涂片检测抗酸菌法和FQ-PCR法对我院临床诊断为肺结核和疑似肺结核病人的168例痰和57例支刷物中的结核分枝杆菌菌体和基因分别同时检测,对两方法检出率进行统计学比较分析。结果痰直接涂片法阳性率为16.7%,FQ-PCR荧光法阳性率为30.4%,二者比较有显著性差异(P<0.01);支刷物涂片阳性率15.8%,TB-PCR荧光量阳性率31.6%,二者比较有差异(P<0.05)。结论FQ-PCR荧光定量法的检出率显著高于涂片法,提示FQ-PCR荧光定量法特异较强、敏感性较高,可提高诊断的阳性率,在结核的诊治中具有较好的应用价值。     

Rapid detection of CYP2C18 genotypes by real-time fluorescence polymerase chain reaction

In man, CYP2C19, a liver enzyme, plays an important role in the metabolism of several drugs. Mutation of the CYP2C19 gene results in a poor metaboliser phenotype. S-Mephenytoin hydroxylation genetic polymorphism is due to two mutations of the CYP2C19 gene, namely CYP2C19*2, located in exon 5, and CYP2C19*3, located in exon 4. CYP2C18 is also polymorphically expressed. The mutant alleles of this enzyme are CYP2C18m1, located in exon 2 and CYP2C18m2, located in the 5'-flanking region. We have developed an allele-specific TaqMan polymerase chain reaction (PCR) assay with which to detect CYP2C18 mutant alleles. This assay combines hybridization of the TaqMan probe and allele-specific amplification primers to the target DNA. The TaqMan probe is labelled with 6-carboxyfluorescein at the 5' end and 6-carboxytetramethylrhodamine together with a phosphate at the 3' end. Genotypes are separated according to the different threshold cycles of the wild type and mutant primers. We applied this procedure to DNA extracted from the blood or saliva of 144 healthy Japanese volunteers. The wt/wt, wt/m1, wt/m2, m1/m1, m1/m2 and m2/m2 genotypes of the CYP2C18 alleles detected by the assay were consistent with the results obtained from restriction enzyme cleavage. In accordance with a previous report, the genotypes of CYP2C18m1 and CYP2C18m2 coincided with those of CYP2C19*3 and CYP2C19*2, respectively. Therefore, detection of CYP2C18 mutant alleles also allows that of CYP2C19 mutant alleles. Among 19 poor metabolisers, eight showed the homozygous CYP2C19*2/CYP2C19*2, two the homozygous CYP2C19*3/CYP2C19*3 and nine the compound heterozygous CYP2C19*2/CYP2C19*3 genotype. We found the allele-specific TaqMan PCR assay rapid, simple and cost-effective, as well as suitable for high-throughput applications in a routine laboratory. This assay allows the fast and reliable detection of inherited disorders that might influence diagnosis and treatment.     

大肠癌组织中环氧化酶2基因表达的实时定量PCR检测

目的探讨环氧化酶2(COX-2)在大肠癌发生和发展中的意义。方法建立实时定量RT-PCR方法,采用LightCycler PCR仪检测了28例大肠癌COX-2 mRNA及内参GAPDH mRNA的表达,以COX-2N=(COX-2拷贝数/GAPDH拷贝数)×103表示COX-2 mRNA表达水平。结果肿瘤组织COX-2 mRNA与正常组织表达水平有极明显差异(P<0.05),转移组织较正常组织COX-2mRNA表达有明显差异(P<0.01)。结论COX-2参与了大肠癌的发生和发展,并起到了重要作用。     

A DNA microarray for the authentication of toxic traditional Chinese medicinal plants

A silicon-based DNA microarray was designed and fabricated for the identification of toxic traditional Chinese medicinal plants. Species-specific oligonucleotide probes were derived from the 5S ribosomal RNA gene of Aconitum carmichaeli, A. kusnezoffi, Alocasia macrorrhiza, Croton tiglium, Datura inoxia, D. metel, D. tatula, Dysosma pleiantha, Dy. versipellis, Euphorbia kansui, Hyoscyamus niger, Pinellia cordata, P. pedatisecta, P. ternata, Rhododendron molle, Strychnos nux-vomica, Typhonium divaricatum and T. giganteum and the leucine transfer RNA gene of Aconitum pendulum and Stellera chamaejasme. The probes were immobilized via dithiol linkage on a silicon chip. Genomic target sequences were amplified and fluorescently labeled by asymmetric polymerase chain reaction. Multiple toxic plant species were identified by parallel genotyping. Chip-based authentication of medicinal plants may be useful as inexpensive and rapid tool for quality control and safety monitoring of herbal pharmaceuticals and neutraceuticals.     

Influence of traditional Chinese anti-inflammatory medicinal plants on leukocyte and platelet functions

The enzymes 5-lipoxygenase and elastase are therapeutic targets in dermatological disorders such as psoriasis. Fifteen extracts from traditional Chinese medicinal plants used to treat topical inflammations were screened for their inhibitory effect on lipoxygenase, cyclooxygenase and elastase activity in intact leukocytes and platelets. Astragalus membranaceus, Forsythia suspensa and Poria cocos inhibited 5-lipoxygenase, with IC50 values of 141, 80 and 141 microg mL(-1), respectively. The latter two species, along with Angelica dahurica and Angelica pubescens, also inhibited elastase (IC50 values of 80, 123, 68 and 93 microg mL(-1), respectively), while A. pubescens, Atractylodes macrocephala, Lentinus edodes, Rehmannia glutinosa and Paeonia lactiflora selectively inhibited 12-(S)-HHTrE production, a valid marker of cyclooxygenase activity. The inhibition of phospholipase A(2) activity by P. cocos is discussed. Dehydrotumulosic and pachymic acids, which have been isolated from P. cocos, were shown to inhibit leukotriene B(4) release. The results indicate that both P. cocos and F. suspensa are potentially valuable species in the management of skin pathologies involving chronic inflammation.     

5种常用清热解毒类中药注射剂不良反应的文献综述

目的 对5种常用中药注射剂不良反应(adverse drug reaction,ADR)的情况调查。方法 收集国内10年来公开发行的医药杂志所报道的中药注射剂不良反应文献,按涉及的器官系统分类进行统计分析。结果 5种中药注射剂的217例ADR中,男性>女性;累及9个类型;以皮肤及附件损害与全身损害为主;62.7％的ADR是在数秒至30 min内发生。结论 临床应高度重视中药注射剂的ADR,确保用药安全。     

孕晚期妇女B族链球菌PCR检测结果分析

目的探讨妊娠晚期孕妇B族链球菌带菌状态的检测方法。方法对在我院产检及分娩的孕妇445例妊娠晚期孕妇,取其阴道下1/3分泌物及肛周分泌物,应用细菌培养及实时PCR进行B族链球菌检测,并观察其妊娠结局。结果445例妊娠晚期孕妇B族链球菌细菌培养阳性17例,实时PCR阳性39例阳性率明显高于细菌培养。B族链球菌阳性孕妇的宫内感染、早产和产后出血发生率明显高于对照组（P〈0.05）。结论实时PCR检测B族链球菌有较高的敏感度和特异性,是妊娠晚期孕妇常规检测B族链球菌感染的首选方法。     

Application of real-time scorpion PCR for authentication and quantification of the traditional Chinese medicinal plant Drynaria fortunei

DNA sequence analysis of the trnl-trnF spacer region and real-time scorpion polymerase chain reaction were exploited for their applications in the differentiation and quantification of the traditional Chinese medicinal plants Drynaria fortunei from five related adulterants: D. mollis, D. quercifolia, D. rigidula, D. sparsisora, and Pseudodrynaria coronans. The data demonstrated that variations in the trnl-trnF spacer regions were very low at the intraspecific level but extremely high at the interspecific level, meaning that they could be easily distinguished at the DNA level. The sequence difference allowed effective and reliable differentiation of D. fortunei from adulterants by real-time scorpion PCR. Furthermore, a quantification methodology was carried out to quantify D. fortunei.     

应用大孔树脂吸附分离技术制备地锦草总黄酮的研究

目的研究大孔树脂DA201和D101富集地锦草总黄酮的工艺条件和参数。方法以总黄酮含量为指标,采用正交试验设计,考察DA201和D101富集地锦草总黄酮的工艺条件。用分光光度法测定总黄酮含量。结果DA201精制的最佳工艺为:90 mL浓度约0.65 mg.mL-1的溶液(pH 7)上柱,吸附流速2 BV.h-1,洗脱剂为40%乙醇(pH 7),洗脱流速2 BV.h-1。D101精制的最佳工艺为:100 mL浓度约0.65 mg.mL-1溶液(pH 7)上柱,吸附流速4 BV.h-1,洗脱剂为40%乙醇(pH 9),洗脱流速2 BV.h-1。通过DA201和D101精制工艺,平均吸附率分别为83.38%和88.57%,平均洗脱率分别为89.28%和87.21%,洗脱液干燥后的总固物中总黄酮平均含量分别为14.48%和20.18%,分别高于原上样液总固物黄酮含量(8.49%和8.71%)。结论DA201和D101对地锦草总黄酮都有良好吸附分离性能,而D101综合性能更好。     

No evidence of involvement of Chlamydia pneumoniae in lung cancer by means of quantitative real-time polymerase chain reaction

Chlamydia pneumoniae, an obligate intracellular pathogen, is well-known as etiological agent of acute respiratory infections; the repeated or prolonged exposure to chlamydial antigens may promote the persistence of C. pneumoniae in the respiratory tract leading to chronic diseases, such as chronic obstructive pulmonary disease and asthma. The predilection of C. pneumoniae to cause respiratory tract infections combined with its persistent nature suggest that it might play a role in lung cancer. The aim of our study is to evaluate the involvement of C. pneumoniae in pathogenesis of lung cancer. We therefore investigated the presence of C. pneumoniae DNA in tumor lung tissues by using real-time PCR assay. Simultaneously, tumor and healthy tissues from the same patient with primary carcinoma lung were analyzed. C. pneumoniae DNA was not detected in a single lung tumor tissue by means of an highly sensitive, and specific real-time PCR assay based on FRET hybridization probes. In conclusion, this study does not support the involvement of C. pneumoniae in the pathogenesis of lung cancer, suggesting that further investigations are needed to clarify other potential causative factors for the development of this malignancy.     

重组酶介导扩增技术与传统聚合酶链反应技术在甲状腺癌DNA甲基化检测中的应用比较

目的 建立重组酶介导的核酸扩增(RAA)技术特异性检测DNA甲基化的新方法并与传统的DNA甲基化特异性PCR(MSP)方法进行比较.方法 选取OXTR基因作为目的基因,提取样品外周血基因组DNA,经亚硫酸氢盐修饰后分别以MSP和RAA技术进行特异性检测DNA甲基化实验.结果 2种技术皆能扩增出OXTR非甲基化条带,而RAA技术成功扩增OXTR甲基化条带.结论 RAA是一种新型的等温体外核酸扩增技术,实现了在37℃恒温下的核酸快速扩增,可成为替代MSP乃至其他PCR实验的新方法.     

Detection of decreased susceptibility to fluoroquinolones in Salmonella spp. by five different methods including real-time polymerase chain reaction (PCR)

Detection of Salmonella spp. isolates showing decreased susceptibility to fluoroquinolones has become important owing to the increasing prevalence of these strains and their association with treatment failure. Nalidixic acid agar dilution, nalidixic acid disk diffusion, MicroScan automated system and real-time polymerase chain reaction (PCR) (LightCycler) followed by melting temperature (Tm) analysis are compared with ciprofloxacin agar dilution as suitable methods to detect decreased susceptibility to fluoroquinolones in 100 Salmonella spp. isolates. Three minor discrepancies were found for nalidixic acid disk diffusion, one minor discrepancy was found for nalidixic acid agar dilution and Tm analysis, and one major discrepancy was found for MicroScan. Nalidixic acid disk diffusion was confirmed as a good screening method. Tm analysis is a rapid and accurate method for detecting decreased susceptibility to fluoroquinolones due to gyrA mutations in Salmonella spp.