实时荧光定量PCR技术的概述及应用

实时荧光定量PCR技术是在传统PCR技术上发展而来的,不仅能判断某一基因的有无,而且还能对其进行定量分析。由于该技术与常规PCR相比,能够进行实时监测、且自动化程度高而被广大研究者青睐。本文对实时荧光定量PCR的原理、数据分析、定量方法、分类以及应用进行了系统而详细地综述。     

茶树小分子量热激蛋白基因CsHSP17.2的克隆与表达分析

[目的]进一步了解茶树小分子量热激蛋白基因CsHSP17.2在逆境胁迫条件下的分子生物学功能.[方法]利用RT-PCR技术从茶树‘迎霜’中克隆得到CsHSP1 7.2基因,运用生物信息学软件分析其核苷酸和编码蛋白,通过Real-timePCR分析其表达模式.[结果]该基因开放阅读框长度为453 bp,编码150个氨基酸,蛋白质相对分子质量为17.2×10a,理论等电点5.56;无信号肽位点,属于非分泌型蛋白;被定位于细胞质中.系统发育树分析表明,茶树CsHSP1 7.2与水稻(GenBank登录号:P27777)和花生(ABC41131)的进化关系较近,属于小分子量热激蛋白基因家族第Ⅰ亚族.qRT-PCR分析发现,茶树CsHSP17.2属于组成型基因;高温(38℃)处理1h能显著提高CsHSP17.2 mRNA的相对表达量(P＜0.05);干旱(100 g·L-1 PEG 6000)、高盐(200 mmol· L-1 NaCl)和外源脱落酸(200 mg· L-1 ABA)处理条件下,该基因的转录水平均出现不同程度的上调.[结论]克隆得到茶树‘迎霜’小分子量热激蛋白基因CsHSP17.2,其在花中表达量最高,且响应高温、干旱、高盐和外源脱落酸胁迫.     

Bt抗虫棉新品系毒蛋白表达差异分析

【目的】本研究旨在探讨部分抗虫棉不抗虫的原因。【方法】通过室内生物测定、卡那霉素、Bt-Cry1Ab/Ac试纸条、PCR分析、Southern blot检测和Bt蛋白含量检测等方法分析了23个抗虫棉新品系的抗虫性。【结果】生测结果表明供试品系间幼虫死亡率差异显著。卡那霉素、Bt-Cry1Ab/Ac试纸条、PCR分析和Southern blot检测结果表明,Bt基因在供试材料中整合并稳定遗传且为单拷贝。RT-PCR结果表明,各试验品系Bt基因相对表达量差异较大,花期叶片Bt基因的相对表达量最高;Bt蛋白检测结果发现抗虫棉Bt蛋白表达量在生育前期大于后期,营养器官中的大于生殖器官,不同年份Bt蛋白含量差异显著。幼虫死亡率与Bt基因相对表达量的相关系数仅为0.1745,与Bt蛋白的相关系数为0.7130,表明Bt蛋白含量越高,抗虫性越好;各组织器官的Bt蛋白与Bt基因的相对表达量相关系数为-0.3659~0.2542,二者表达趋势不一致,表明遗传背景对抗虫棉Bt蛋白的表达具有一定影响。且Bt基因可能在转录后受到调控导致Bt蛋白含量发生变化,从而影响了抗虫棉的抗虫性。【结论】这些结果有望为抗虫棉育种提供参考。     

数字PCR技术在动物疫病检测中的应用

为了支持动物疫病流行地区的控制及消除计划,以及在动物疫病非流行地区进行有效筛查,必须使用更加准确灵敏的诊断手段。数字PCR可实现绝对定量及痕量核酸的检测,与传统检测方法相比具有不依赖参考物或标准品、对抑制剂具有较高耐受性、灵敏度和精准度更高的优点。数字PCR作为一种定量分析的核酸检测新方法,在动物疫病检测中得到了应用,围绕数字PCR技术的原理、应用、存在的问题及发展趋势等进行综述及分析,为数字PCR在动物疫病检测中的进一步应用提供参考。     

A serological and molecular study on Francisella tularensis in rodents from Hamadan province,Western Iran

Introduction and purposeTularemia is a zoonotic disease, the most important hosts of which are rodents. Endemic regions and reservoirs of F. tularensis are not well-researched areas in Iran. The present study aimed to study F. tularensis infection in the rodent populations of western Iran.Materials and methodsSamples were collected in different areas of Kabudar Ahang County in Hamadan province (west of Iran) from 2014 to 2017. Tularemia serological and molecular tests were conducted using the tube agglutination test and Real-time PCR method tracking the ISFtu2 gene. Positive serum samples were evaluated for cross-reactivity with brucellosis.ResultsA total of 433 rodents, collected from 33 localities, were included in the study. The most abundant species belonged to the Persian jird (Meriones persicus; 75.5%), and Libyan jird (Meriones libycus; 10.1%). Among the studied samples, three (0.74 %) were seropositive and five (1.15%) were PCR positive. Seropositive samples were two M. persicus and one M. libycus, and PCR positive rodents were four M. persicus and one M. vinogradovi. Tularemia seropositive samples showed no cross-reactivity with brucellosis.ConclusionGiven the presence of infection in rodents with tularemia agent in the studied area, it is crucial to elucidate the risks of rodent exposure to tularemia for physicians, health personnel and the general population.     

Serological and molecular detection of infection with Mycobacterium leprae in Brazilian six banded armadillos (Euphractus sexcinctus)

Leprosy was recognized as a zoonotic disease, associated with nine-banded armadillos (Dasypus novemcinctus) in the Southern United States of America in 2011. In addition, there is growing evidence to support a role for armadillos in zoonotic leprosy in South America. The current study evaluated twenty specimens of the six-banded armadillo (Euphractus sexcinctus), collected from rural locations in the state of Rio Grande do Norte (RN), Brazil for evidence of infection with Mycobacterium leprae. Serum was examined using two "in-house" enzyme-linked immunosorbent assays (ELISAs) and via two commercially available (ML flow and NDO-LID®) immunochromatographic lateral flow (LF) tests, for detection of the PGL-I and/or LID-1 antigens of the bacterium. The presence of M. leprae DNA in liver tissue was examined using the multi-copy, M. leprae-specific repetitive element (RLEP), as target in conventional and nested PCR assays. Molecular and anti-PGL-I-ELISA data indicated that 20/20 (100 %) of the armadillos were infected with M. leprae. The corresponding detection levels recorded with the LF tests were 17/20 (85 %) and 16/20 (85 %), for the NDO-LID® and ML flow tests, respectively. Our results indicate that, in common with D. novemcinctus, six banded armadillos (a species hunted and reared as a food-source in some regions of Brazil, including RN), represent a potential reservoir of M. leprae and as such, their role in a possible zoonotic cycle of leprosy within Brazil warrants further investigation.     

Molecular survey of Dirofilaria species in stray dogs,red foxes and golden jackals from Vojvodina,Serbia

Cardiopulmonary dirofilariosis in dogs and other carnivores is caused by Dirofilaria immitis, while Dirofilaria repens usually causes a subcutaneous infection. The importance of red foxes and golden jackals in the epidemiology of dirofilariosis remains unknown. Thus, the aim of this study was to conduct a cross-sectional molecular survey of Dirofilaria species in stray dogs, red foxes and golden jackals from the endemic region of Vojvodina, Serbia, in order to determine and update data on their prevalence and provide insight into the epidemiological importance of wild canids.A total of 59 blood samples from stray dogs, 94 from red foxes and 32 from golden jackals were collected and screened by real-time PCR targeting a 115-bp fragment of the mitochondrial 12S gene of filarioids and by conventional PCR assay targeting a 484–524-bp fragment of 5.8S-ITS2-28S locus of filarioids.The cross-sectional molecular survey detected the filarioid mitochondrial 12S gene fragment in stray dogs (27.1 %), red foxes (8.5 %) and golden jackals (6.3 %) in the same endemic region of Vojvodina, Serbia. Only D. immitis was detected in stray dogs, while both D. immitis and D. repens were detected in populations of red foxes and golden jackals. These results outline a possible interaction of D. immitis infection between the dog population and the wild canid populations, while D. repens was found to circulate mostly in golden jackals and red foxes populations.     

Death of a neonatal lamb due to Clostridium perfringens type B in New Zealand

ABSTRACT

Case history and clinical findings: A flock of 20 sheep was kept within three paddocks on a single property. None of the animals in the flock had been vaccinated against any disease for at least three years. Abdominal bloating and haemorrhagic diarrhoea were observed in Lamb 1 at 24 hours-of-age. The lamb subsequently died within an hour of the onset of clinical signs. Lamb 2 was 3-days-old when observed to be recumbent with opisthotonus. The lamb was treated with dextrose, vitamins B1 and B12, and penicillin G, but died 4 hours later.

Pathological findings: Examination of Lamb 1 revealed markedly increased gas within the peritoneum and within dilated loops of intestine. The intestines were dark red and contained large quantities of haemorrhagic fluid. Histology of the intestines revealed peracute mucosal necrosis with minimal accompanying inflammation. The intestinal lumen contained cell debris, haemorrhage, and myriad large Gram-positive bacilli. The intestines of Lamb 2 did not appear bloated or reddened. However, multiple fibrin clots were visible within the pericardial sac. Histopathological examination revealed small foci of necrosis within the mucosa of the distal intestine. The necrotic foci were often associated with large numbers of large Gram-positive bacilli.

Immunohistochemsitry and molecular biology: Intestinal samples from Lamb 1 were processed for Clostridium perfringens immunohistochemistry, which revealed large numbers of intralesional, positively immunostained rods. Fragments corresponding to the expected sizes for genes encoding alpha, beta, and epsilon C. perfringens typing toxins were amplified by PCR from DNA extracted from formalin-fixed sections of intestine.

Diagnosis: Lamb dysentery due to C. perfringens type B.

Clinical relevance: C. perfringens bacteria have a worldwide distribution, but disease due to C. perfringens type B has only been diagnosed in a small number of countries and has never been reported in New Zealand or Australia. C. perfringens type B produce both beta toxin and epsilon toxins, therefore both haemorrhagic enteritis and systemic vascular damage can develop. As many animals are exposed to C. perfringens without developing disease, there must be additional unknown factors that resulted in disease in these particular sheep. Vaccines that specifically protect against C. perfringens type B are available and may be recommended for use in smaller non-commercial flocks, as in the present case.     

Serum amyloid A (SAA) mRNA expression in chicken and quails in response to bacterial stress

Monitoring of acute phase proteins such as serum amyloid A at gene expression level may provide quick information about immune status of the host and its susceptibility towards common infections. Present study was carried out to evaluate and compare the mRNA expression of SAA gene in Rhode Island Red chicken (RIR) and Japanese quails using real time PCR analysis in response to inactivated Salmonella gallinarum culture. The results showed that expression of SAA gene was approximately 17–33 folds higher in case of birds administered with bacterial culture when compared to un-inoculated controls and expression was higher and quicker in case of quails than RIR chicken. The SAA genes from chicken and quail were cloned and upon sequence analysis it was observed that deduced amino acid sequence of SAA from chicken and quails were having approximately seven percent variation which might have significance in function of this protein in these species.     

Outbreak of anaplasmosis associated with novel genetic variants of Anaplasma marginale in a dairy cattle

During early lactation, dairy cows may present a transient immunosuppressive state and develop anaplasmosis caused by Anaplasma marginale. In this study, clinical anaplasmosis in dairy cattle in the Thrace region of Turkey was investigated with respect to within-herd prevalence, vertical transmission, and genetic diversity. In March and September 2015, thirty lactating cows showed primary clinical signs of anaplasmosis, including fever, anaemia, decreased milk yield, anorexia, and laboured breathing. Symptoms disappeared in most cows after administration of long-acting oxytetracycline, but nine of them (30%) died. Following diagnosis based on clinical signs, microscopy and molecular findings, blood samples were collected from apparently healthy lactating cows (n = 184), pregnant heifers (n = 39) and newborn calves (n = 24). DNA was extracted from each sample and analyzed for the presence of major surface proteins (MSPs) of A. marginale, followed by sequencing to assess diversity of isolates. Microscopic examination of erythrocytes revealed A. marginale inclusion bodies in symptomatic cows. Examination of thin blood smears showed 3.8% of the lactating, clinically asymptomatic, cows to be infected with A. marginale, while nPCR detected 31.0% positive. A. marginale infection was not detected in pregnant heifers by either method. Congenital infection was found in one calf by nPCR. This is the first report of transplacental transmission of A. marginale in Turkey. The MSP4 sequence analyses showed high genetic diversity among the isolates, presenting 97.6-99.6% homology at the amino acid level. The sequences of MSP1a amplicons revealed genetic diversity providing three new tandem repeats.