环介导等温扩增联合横向流动试纸条可视化检测海豚链球菌方法的建立

海豚链球菌(Streptococcus iniae)是一种革兰氏阳性球菌,呈β溶血,可感染多种淡水和海洋鱼类。本研究利用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)进行核酸扩增,通过横向流动试纸条方法 (lateral flow dipstick,LFD)实现检测,建立了一种可应用于海豚链球菌快速检测的LAMP-LFD技术。该技术以海豚链球菌促旋酶B亚单位(gyrase subunit B,gyr B)基因为检测靶标,设计3对引物进行由生物素标记的LAMP扩增反应,产物经异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记的探针杂交后,在LFD上完成检测。经优化的核酸扩增最适条件为65℃反应30 min,在此条件下,阳性扩增起始时间与模板浓度之间呈典型的线性相关性。从核酸扩增反应开始到LFD显色,整个检测时程只需40 min左右,比常规PCR技术缩短约2 h。LAMP-LFD能特异性地检出海豚链球菌,针对病原纯培养物的检测灵敏度为8.70×101cfu/m L,是LAMP检测的10倍、常规PCR检测的100倍。以灵敏度浓度(8.70×101cfu/m L)的海豚链球菌基因组DNA为模板进行的LAMP-LFD结果显示该方法具有良好的重复性。针对人工污染花鲈(Lateolabrax japonicus)肝组织的检测灵敏度为4.35×103cfu/m L,同样为常规PCR检测方法的100倍。利用本方法可成功从患病花鲈的组织样品中检测出海豚链球菌,检测结果与常规的细菌分离鉴定方法结果一致。因此,利用LAMP-LFD能特异、准确、高效地检测出海豚链球菌,而且操作简单、费用低、耗时短,有望成为海豚链球菌的常规检测方法。     

Detection of canine microalbuminuria using semiquantitative test strips designed for use with human urine

Background — Commercial testing for microalbuminuria in human urine is often performed with point-of-care semiquantitative test strips followed by quantitative testing when indicated. An ELISA that quantifies canine urine albumin concentration has been developed, but semiquantitative test strips for use in the dog are not available.
Objective — The purpose of this study was to prospectively determine the concordance of canine urine albumin concentrations measured by a commercial human test strip and by ELISA.
Methods — Urine samples were obtained from 67 dogs evaluated for a variety of clinical conditions. Dipstick urinalyses were performed on all samples; clinician discretion determined method of urine collection and performance of urine sediment examination and/or urine culture. Urine albumin concentration was determined using test strips (Clinitek Microalbumin, Bayer Corporation, Elkhart, Ind, USA), and results were compared with those obtained by ELISA.
Results — The Clinitek strips correctly determined albumin concentration in 42 of 67 (63%) urine samples tested. Concordance was lowest (48%) for dogs with microalbuminuria (10–300 μg/mL by ELISA). Clinitek strip sensitivity and specificity for correct identification of microalbuminuria were 48% and 75%, respectively. Concordance was lower in dogs with urinary tract infection or hematuria and in samples collected by catheterization. Sensitivity and specificity for correct identification of microalbuminuria after exclusion of dogs with urinary tract infection or hematuria were 59% and 83%, respectively.
Conclusion — These results suggest that the Clinitek strips lack sufficient concordance with results obtained by ELISA to be reliable screening tests for microalbuminuria in the dog. A reliable semiquantitative point-of-care test for canine urine albumin concentrations below those detected by standard urine dipsticks is still needed.     

胶体染料试纸条法快速诊断家畜血吸虫病的研究

为了适应现场家畜血吸虫病的筛查需要,设计了简便快速的胶体染料试纸条诊断技术用于检测家畜血清中的抗血吸虫抗体,以该方法检测血吸虫病牛、猪血清83份,其结果为阳性检出率100%,健康牛、猪血清73份的检测结果为阴性符合率分别为95.3%和94.0%,与感染肝片形吸虫的牛、羊血清16份的交叉反应率为18.8%.表明本方法敏感性高,特异性较强.     

Comparison of Multistix PRO dipsticks with other biochemical assays for determining urine protein (UP), urine creatinine (UC) and UP:UC ratio in dogs and cats

BACKGROUND: Urine protein: urine creatinine (UP:UC) ratio determined from the quantitative measurement of protein and creatinine in a single urine sample is the best feasible assessment of clinically significant proteinuria in dogs and cats. A dipstick that measures urine protein, urine creatinine, and UP:UC ratio has been used in human medicine and could have application for veterinary practice. OBJECTIVE: The objective of this study was to compare the Multistix PRO dipstick (Bayer Corporation, Elkhart, IN, USA) to other biochemical methods for determination of urine protein and creatinine, and UP:UC ratio in canine and feline urine. METHODS: A complete urinalysis, including sulfosalicylic acid (SSA) precipitation, was performed on urine samples submitted to our laboratory between February and April 2003 from 100 dogs and 49 cats. Urine protein and creatinine concentrations were determined by the Multistix PRO dipstick using a Clinitek 50 analyzer (Bayer) and compared with the results of SSA precipitation and quantitative biochemical analysis. The UP:UC ratios from the dipstick results (calculated by the Clinitek 50 and also manually) were compared with those calculated from quantitative values. Pearson product-moment correlation analysis and diagnostic sensitivity and specificity (using quantitative results as the gold standard) were determined. RESULTS: For both canine and feline urine, protein and creatinine concentrations determined by the Multistix PRO correlated closely with quantitative concentrations for protein (dogs r = .78, P = .0001; cats r = .87, P = .0001) and creatinine (dogs r = .78, P = .0001; cats r = .76, P = .0001). The Multistix PRO was more sensitive and less specific than SSA precipitation for diagnosing clinically significant proteinuria. UP:UC ratios obtained by manual calculation of dipstick results correlated best with quantitative UP:UC ratios in dogs, and had higher specificity but lower sensitivity for the diagnosis of proteinuria. In cats, UP:UC ratios determined by the dipstick method did not correlate (r = -.24, P = .0974) with quantitative values. CONCLUSIONS: The Multistix PRO, with manual calculation of UP:UC, may be a good alternative for the diagnosis of clinically significant proteinuria in dogs, but not cats. Dipstick creatinine concentration should be considered as an estimate.     

芋疫霉重组聚合酶扩增结合侧流层析试纸条快速检测方法的建立及应用

为建立芋疫霉Phytophthora colocasiae快速准确的分子检测方法,基于Ypt1基因特异序列,设计芋疫霉的特异性引物与探针,建立一种快速、准确、可视化的芋疫霉重组聚合酶扩增结合侧流层析试纸条（recombinase polymerase amplification-lateral flow dipstick,LFD-RPA）检测方法,对该检测方法进行优化,评估其特异性与灵敏度,并对田间疑似样品进行检测。结果表明,优化后的芋疫霉LFD-RPA检测方法最适反应条件为39℃恒温反应30 min。LFD-RPA检测方法能够特异性地检测出芋疫霉,而对其他卵菌近缘种和常见植物病原真菌均未检出,且该检测方法对芋疫霉DNA的检测灵敏度达到1 pg/μL。对田间带病组织检测发现,LFD-RPA检测方法能够快速准确地从田间自然发病植株中检测出芋疫霉。表明本研究所建立的芋疫霉LFD-RPA快速可视化检测方法特异性好、灵敏度高、简单快捷,可用于芋疫病的田间快速诊断。     

Evaluation of the effect of urine dip vs urine drip on multi‐test strip results

Standard operating procedures, including World Health Organization guidelines for packed cell volume, are established for in‐clinic laboratory tests. No independent, evidence‐based guidelines exist for dipstick urinalysis; however, manufacturer's instructions state to dip the stick into urine. In veterinary medicine, small volume urine samples could preclude dipping; therefore, a single drip per pad from a pipette or syringe is often performed. This study aimed to examine the differences between these two urine application methods prior to analysis, with the hypothesis that the method type would not effect on test results of dipstick analysis. To standardize the strip analysis method, a Siemens Clinitek Status + analyzer was used with Multistix10SG dipsticks. Three investigators tested urines from 53 dogs with a range of diseases by both methods. Results were assessed for the degree of agreement between the methods and within method variability. Overall, the agreement between methods was high. Within each method, the drip method variability was higher than that of the dip method (P = 0.012). Disagreements between methods were present, with pH and blood having the lowest agreement levels. Glucose was more likely to be positive on the drip compared with the dip methodology. This study demonstrates potential clinically relevant differences between the two methods and a higher level of variability with the drip methodology. Therefore, while the drip method could be used for practical reasons (eg, low sample volumes), this study supports the manufacturer's recommended method of dipping the dip stick into urine rather than dripping urine onto each pad with a pipette or syringe.     

樱桃灰霉病菌LFD-RPA快速检测方法的建立

 灰霉病是樱桃的主要病害,由灰葡萄孢(Botrytis cinerea)侵染所致。该病菌可为害樱桃的花、叶、果实等多个部位,尤其在樱桃生长中后期及贮藏期发病率极高,危害严重。依据樱桃灰霉病菌内转录间隔区(ITS)序列设计引物,建立了樱桃灰霉病菌的重组酶聚合酶扩增结合侧流层析试纸条(LFD-RPA)检测方法。结果表明,该方法检测条件为37℃扩增30 min,能特异性地检测灰葡萄孢,灵敏度为100 fg·μL^-1,略低于常规PCR(10 fg·μL^-1)和荧光定量PCR(7.43 fg·μL^-1);检测方法用时短,PRA扩增仅需30 min,LFD检测仅需10 min。本研究建立的快速检测方法可用于樱桃灰霉病菌的实时监测及病害的田间快速诊断。