A rapid method for extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis

Extraction of high-quality genomic DNA fromGossypium (cotton) species is difficult due to high levels of polysaccharide, oxidizable quinones, and other interfering substances. We describe a procedure that consistently permits isolation of cotton genomic DNA of satisfactory size and quality for RFLP and PCR analysis, as well as for most routine cloning applications. Several antioxidants, phenol-binding reagents, and phenol oxidase inhibitors are used throughout the procedure, and most polysaccharides are eliminated early in the procedure by isolation of nuclei.     

Evaluation of housekeeping genes as references for quantitative real-time PCR analysis of European eel,Anguilla anguilla

Eels are important aquaculture species for which an increasing number of reference genes are being identified and applied. In this study, five housekeeping genes [RPL7 (ribosomal protein L7), 18 S (18 S ribosomal RNA), EF1A (elongation factor 1α), ACTB (β-actin) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase)] were chosen to evaluate their reliability as reference genes for quantitative real-time PCR (qPCR) for the study of Anguilla anguilla. The expression of the selected genes in different eel tissues was determined using qPCR at different growth stages or upon challenge by Anguillid herpesvirus (AngHV), and the expression levels of these genes were then compared and evaluated using the geNorm and NormFinder algorithms. Then, RefFinder was used to comprehensively rank the examined housekeeping genes. Interestingly, the expression of the evaluated housekeeping genes exhibited tissue-dependent and treatment-dependent variations. In different growth periods A. anguilla tissues, the most stable genes were the following: ACTB in mucus; 18 S in skin and kidney; RPL7 in muscle, gill, intestine and brain; EF1A in heart and liver; and GAPDH in spleen. In contrast, in AngHV-challenged A. anguilla tissues, the most stable genes were the following: 18 S in mucus; RPL7 in skin, gill, heart, spleen, kidney and intestine; EF1A in muscle and liver; and ACTB in brain. Further comparison analysis indicated that the expression of RPL7 and EF1A was stable in multiple A. anguilla tissues in different growth periods and in eels challenged by AngHV. Nonetheless, the expression level of GAPDH in eel tissues was lower, and it was unstable in several tissues. These results indicated that the selection of reference genes for qPCR analysis in A. anguilla should be made in accordance with experimental parameters, and both RPL7 and EF1A could be used as reference genes for qPCR study of A. anguilla at different growth stages or upon challenge by AngHV. The reference genes identified in this study could improve the accuracy of qPCR data and facilitate further studies aimed at understanding the biology of eels.     

PCR-mediated screening and labeling of DNA from clones

A simplified and economical protocol for DNA library screening and nonradioactive labeling is described. Bacterial clones are lysed in 1% of Triton X-100 and subjected to polymerase chain reaction in the presence of digoxigenin-11-dUTP to screen and simultaneously to label the DNA inserts. Bacteriallysates are stable in storage at −20°C and can be used repeatedly for PCR-mediated labeling. In this protocol, very low concentrations of dNTP, digoxigenin-dUTP, and primers are used in combination with a reduced reaction volume. This will considerably reduce the expense of screening and labeling bacterial clones and facilitate the exchange of DNA probes among laboratories.     

The progress of DNA analyzing techniques and its impact on plant molecular systematics

Recent advances in manipulating nucleic acids have opened a new research field called plant molecular systematics. This short review provides an overview of molecular techniques which have been used in the analysis of DNA molecules for the study of plant systematics, with a special emphasis on PCR. The early application of DNA analysis, DNA/DNA hybridization, has not become popular with plant systematists, because of several disadvantages inherent in the method. The survey of restriction fragment length polymorphisms (RFLPs), on the contrary, has become one of the preferred methods used by plant molecular systematists, since the method is relatively easy to perform. Although unambiguous data can be obtained by both long-range restriction mapping and nucleotide sequencing, these approaches may have limited use in plant molecular systematics because of their laborious experimental procedures relying on conventional molecular cloning techniques. To date, PCR based analyses of the DNA molecule seem to be the most suitable experimental approach for plant molecular systematics. Several advantages of the method have changed both the quality and quantity of the DNA data. Further application of PCR to plant molecular systematics will open up a new era in the field. The present paper is based on the contribution which was read in a symposium entitled “Organellar DNA Variations in Higher Plants and their Taxonomic Significance”, at the 50th Annual Meeting of the Botanical Society of Japan in Shizuoka on October 2, 1990, under the auspices of the Japan Society of Plant Taxonomists.     

笼养蓝马鸡取食高度偏好

笼养鸟类取食高度对其自然行为表达和动物福利至关重要。然而,相关研究却少见报道。本文以我国二级重点保护野生动物——蓝马鸡(Crossoptilon auritum)为研究对象,观察笼养状态下其对不同高度取食槽内放置的种子或蔬菜的取食次序,并使用回归分析建立取食高度与偏好值的数学模型。结果发现,笼养蓝马鸡取食种子类食物(玉米粒)最佳取食高度为0cm,并且采食偏好随采食槽高度升高而下降(y=-0.564x+43.146,R²=0.946,y为采食偏好值,x为采食槽高度);油麦菜(Lactuca sativa)的最适取食高度范围为15～25 cm,随着取食槽高度的升高,偏好值先上升,至25 cm处后下降(y=-0.014x2+0.543x+26.487,R²=0.952,y为采食偏好值,x为采食槽高度)。当取食槽高度在65 cm及以上时,蓝马鸡拒食率上升(拒食率≥38.9%)。成年蓝马鸡体高对取食高度影响不明显。研究结果可为笼养蓝马鸡饲养管理提供参考。     

基于双重芯片式数字PCR快速鉴定KPC型碳青霉烯耐药肺炎克雷伯菌的方法

【背景】由于碳青霉烯类药物的泛用和滥用,致使肺炎克雷伯菌碳青霉烯耐药株与日俱增,产碳青霉烯酶是肺炎克雷伯菌对碳青霉烯类药物耐药的主要原因。目前对肺炎克雷伯菌碳青霉烯耐药株的检测方法存在费时费力、特异性差、灵敏度低等问题。【目的】建立一种能同时检测肺炎克雷伯菌和碳青霉烯酶基因bla_KPC的双重芯片式数字PCR方法。【方法】依据肺炎克雷伯菌的特有基因yhaI和碳青霉烯耐药基因bla_KPC保守序列设计特异性引物和探针,确定双重芯片式数字PCR同时对yhaI和bla_KPC两个基因核酸浓度绝对定量的检测范围、检出限和最佳实验体系,并进行方法特异性、灵敏度、重复性分析及临床菌株的检测。【结果】双重芯片式数字PCR检测灵敏度比双重实时荧光定量PCR提高了约1.5个数量级,在两基因同时检出的情况下,最低检出限分别为3.74 copies/μL (yhaI基因)和1.93 copies/μL (bla_KPC基因);优化后的双重芯片式数字PCR对参考菌株检测特异性的结果与双重实时荧光定量PCR结果一致;利用优化后的双重芯片式数字PCR方法共检测58株临床菌株,其中肺炎克雷伯菌43株,属肺炎克雷伯菌且含有bla_KPC基因的菌株13株,这与质谱及耐药谱检测结果一致。【结论】利用双重芯片式数字PCR技术建立了产KPC型碳青霉烯酶肺炎克雷伯菌的绝对定量检测方法。该方法特异性强、灵敏度高、准确度好,可用于检测具有碳青霉烯酶基因bla_KPC的肺炎克雷伯菌的核酸检测和定量分析,也为产其他类型碳青霉烯酶的病原菌检测提供了新的技术参考。     

Palynological and palaeobotanical investigations in the area of the post-medieval Helsinki Old Town

Pollen, phytolith, and plant macrofossil analyses were carried out on the cultural layers of Helsinki Old Town excavated by the Helsinki City Museum and dated to the 15–17th centuries. Cultural mineral soil layers, a former ditch, a well, one waste pit, one waste heap and three house areas were investigated. The results show that-within certain limits-pollen in the mineral soil reflects the cultural changes of the local settlement from rural to urban. Among the main anthropogenic indicators, the high NAP (non-arboreal pollen) frequencies, the low pollen concentration values, and especially the high frequencies of Cichoriaceae (Liguliflorae) pollen should be emphasized. The macrofossil data were used to determine the species list of the herb pollen and to make a more detailed reconstruction of the local vegetation at Helsinki Old Town.     

乳腺良恶肿瘤患者超声弹性成像定量参数与临床分期、病理分子分型的相关性分析

摘要 目的：探讨乳腺良恶肿瘤患者超声弹性成像定量参数与临床分期、病理分子分型的相关性。方法：选择2020年1月至2022年12月来我院诊治的乳腺肿块患者85例,均行超声弹性成像检查,分析85例乳腺肿块患者的病理检查结果,对比良恶性肿瘤患者的弹性成像参数,对弹性应变率、直径变化率、面积比及三者联合绘制ROC曲线,分析不同乳腺肿瘤患者临床分期的弹性成像参数,分析乳腺肿瘤患者病理分子分型的弹性成像参数。结果：85例乳腺肿块患者中,良性肿块35例,恶性肿块50例。恶性组的弹性应变率、肿块直径、直径变化率、肿块面积、面积比明显较良性组低（P<0.05）。面积比ROC曲线AUC为0.580,以1.73为临界值,乳腺恶性肿瘤的诊断灵敏度为73.5 %,特异度为38.5 %;直径变化率ROC曲线AUC为0.630,以0.28为临界值,诊断灵敏度为75.5 %,特异度为47.5 %;弹性应变率ROC曲线AUC为0.790,以15.2 cm²为临界值,诊断灵敏度为64.5 %,特异度为83.5 %,以三者联合绘制ROC曲线,AUC为0.920,诊断灵敏度为82.5 %,特异度为92.5 %。乳腺恶性肿瘤患者TNM分期Ⅰ、Ⅱ、Ⅲ、Ⅳ期者的弹性应变率、肿块直径、直径变化率、肿块面积、面积比对比有统计学意义（P<0.05）;其中Ⅳ期者的弹性应变率、肿块直径、直径变化率、肿块面积、面积比明显较Ⅲ、Ⅱ、Ⅰ期者高,Ⅲ期者明显较Ⅱ、Ⅰ期者高,Ⅱ期者明显较Ⅰ期高。乳腺恶性肿瘤患者Luminal A型者、Liminal B型者、Her2过表达型者、基底样型者的弹性应变率、肿块直径、直径变化率、肿块面积、面积比对比有统计学意义（P<0.05）;其中Liminal B型者的弹性应变率、肿块直径、直径变化率、肿块面积、面积比明显较Luminal A型者、Her2过表达型者、基底样型者高,Her2过表达型者明显较Luminal A型者、基底样型者高（P均<0.05）,Luminal A型者与基底样型者对比无统计学意义（P>0.05）。结论：超声弹性成像可用于乳腺良恶肿瘤的诊断,超声弹性成像定量参数可用于恶性乳腺肿瘤临床分期、Liminal B型、Her2过表达型的判断。     

食管癌患者放射治疗中照射体积和时间与外周血淋巴细胞绝对值的相关性研究

摘要 目的：探究照射体积和时间与食管癌患者外周血淋巴细胞绝对值的相关性。方法：本研究方案将纳入2019年1月~2019年12月蚌埠医学院第一附属医院放疗科收治的放疗或同步放化疗食管癌患者84例,其中单独放疗患者24例,同步放化疗患者60例,采用血液细胞分析仪测定患者放疗期间每周复查外周血白细胞（WBC）、中性粒细胞（N）、淋巴细胞（L）、血红蛋白（HB）及血小板（PLT）计数等指标。Pearson相关性分析照射时间、剂量及体积与外周血指标之间的相关性。结果：食管癌放疗患者,包括同步放化疗及单纯放疗亚组,在治疗1-6周,照射时间与外周血指标均无相关性（P>0.05）。但在放疗第5-6周,患者放疗剂量与WBC、N、L、HB呈负相关（P<0.05）,同步放化疗亚组患者照射剂量与WBC、N、L、HB呈负相关（P<0.05）。在治疗1-4周,不同照射剂量下各梯度照射剂量对应照射体积与外周血指标均无相关性（P>0.05）。但在第5-6周时,患者不同梯度照射剂量下各照射体积与WBC、N呈负相关（P<0.05）,同时在20Gy-60Gy照射剂量,尤其20Gy和30Gy照射剂量下照射体积与L呈负相关（P<0.05）。同步放化疗亚组患者不同照射剂量下各照射体积与WBC、N呈负相关（P<0.05）,同时在20Gy-60Gy照射剂量下照射体积与L呈负相关（P<0.05）,而且在60Gy照射剂量下照射体积与HB呈负相关（P<0.05）。结论：放疗患者特别是同步放化疗亚组患者照射体积、照射剂量与食管癌患者外周血淋巴细胞计数成负相关,基线淋巴细胞与食管癌患者外周血淋巴细胞计数成正相关,而照射时间与食管癌患者外周血淋巴细胞计数无相关性。     

Epidemiology of apple proliferation (AP) in northwestern Italy: evaluation of the frequency of AP-positive psyllids in naturally infected populations of Cacopsylla melanoneura (Homoptera: Psyllidae)

Adults of Cacopsylla melanoneura, vector of the apple proliferation (AP) phytoplasma, were collected every 2 weeks from January until May in 2000 and 2001 by the beating tray method in eight apple orchards of the Aosta Valley (northwestern Italy). Total DNA was extracted from batches of five insects and amplified with the universal phytoplasma primers P1/P7 in direct PCR. A nested PCR assay was then performed on P1/P7 amplicons using the primers fO1/rO1, specific for the AP‐ phytoplasma group. The digestion of fO1/rO1 amplicons with Ssp I restriction endonuclease confirmed that C. melanoneura adults harboured the AP phytoplasma. The data obtained with PCR were used to estimate the proportion of AP‐positive insects in over wintered and offspring adults. Percentages of AP‐positive insects of 3.6% and 0.8% were estimated in 2000 among over wintered and offspring psyllids respectively. In 2001 only the over wintered insects were found infected, with an estimated proportion of 2.8%. The seasonal abundance of the vector was measured using yellow sticky traps. C. melanoneura was always present at a low population level, and the highest density was recorded from mid‐February until mid‐March in both years. The results show that the overwintered population is higher and spends a longer period in apple orchards, suggesting the crucial role of the overwintered adults in vectoring AP.