高致病性猪繁殖与呼吸综合征病RT-PCR快速鉴别诊断方法的建立与临床应用

根据高致病性猪繁殖与呼吸综合征病毒(PRRSV)Nsp2基因的缺失信息,设计了3条特异性引物,以含高致病性PRRSV Nsp2基因的质粒pMDNSP2及普通PRRSV VR2332株RNA为模板,建立了快速诊断高致病性PRRSV RT-PCR方法.通过对临床组织病料的总RNA进行不同稀释倍数检测,结果表明该方法能从0.265 pg的总RNA中检测到PRRSV的基因,说明敏感性高.用该方法对猪瘟病毒(CSFV)、Ⅱ型圆环病毒(PCV-2)、伪狂犬病病毒(PRV),链球菌(Streptococcus)、副猪嗜血杆菌(Haemophilus parasuis)和大肠杆菌(Escherichia coli)同条件检测,结果都为阴性.进一步对36份疑似高致病性PRRSV临床组织病料细胞培养物、2株PRRSV商品活疫苗以及52个猪场所送检的184份临床样品进行了检测应用,结果36份疑似高致病性PRRSV临床组织病料的细胞培养物有5份样品为阳性且都为高致病性PRRSV,2株PRRSV商品活疫苗为普通PRRSV,52个猪场中有42个猪场(123份样品)呈阳性,其中只有1份为普通PRRSV.实验表明该方法能够准确地鉴别诊断高致病性PRRSV和普通PRRSV,且具有快速,敏感和特异的特点,具有临床实用性.     

Evaluation of nine sets of PCR primers in the RNA dependent RNA polymerase region for detection and differentiation of members of the family Caliciviridae, Norwalk virus and Sapporo virus

Norwalk virus and Sapporo virus were approved as type species of the genus "Norwalk-like viruses" and the genus "Sapporo-like viruses," respectively, in the family Caliciviridae. A total of 116 stool specimens containing Norwalk virus (NV) or Sapporo virus (SV) were tested by RT-PCR and Southern hybridization to evaluate nine sets of PCR primers and seven internal oligonucleotide probes in the RNA dependent RNA polymerase region of NV and SV for detection and differentiation of viruses in the NV and SV. Fifty-five stool samples were collected from 11 outbreaks of NV and/or SV gastroenteritis in an infant home, where residents were infants under 2 years of age, in Sapporo, Japan. Sixty specimens were obtained in Sapporo from sporadic cases in children, mainly under 6 years of age, of acute gastroenteritis due to small round structured viruses detected by EM. There is no single primer pair to detect all NV and SV, and at least three primer pairs, G1 set, G2 set and Sapp35/Sapp36, are required to detect viruses in the NV and SV clades. Many NV and SV strains were successfully classified into one of the NV/genogroup I, NV/genogroup II and SV by single-round RT-PCR and Southern hybridization. The new detection method for SV reported in this study combined with those for NV previously reported may elucidate the importance of Norwalk virus and Sapporo virus as a cause of viral gastroenteritis in all age groups in the world.     

The Development and Application of the Two Real-Time RT-PCR Assays to Detect the Pathogen of HFMD

Large-scale Hand, Foot, and Mouth Disease (HFMD) outbreaks have frequently occurred in China since 2008, affecting more than one million children and causing several hundred children deaths every year. The pathogens of HFMD are mainly human enteroviruses (HEVs). Among them, human enterovirus 71 (HEV71) and coxsackievirus A16 (CVA16) are the most common pathogens of HFMD. However, other HEVs could also cause HFMD. To rapidly detect HEV71 and CVA16, and ensure detection of all HEVs causing HFMD, two real-time hybridization probe-based RT-PCR assays were developed in this study. One is a multiplex real-time RT-PCR assay, which was developed to detect and differentiate HEV71 specifically from CVA16 directly from clinical specimens within 1–2 h, and the other is a broad-spectrum real-time RT-PCR assay, which targeted almost all HEVs. The experiments confirmed that the two assays have high sensitivity and specificity, and the sensitivity was up to 0.1 TCID₅₀/ml for detection of HEVs, HEV71, and CVA16, respectively. A total of 213 clinical specimens were simultaneously detected by three kinds of assays, including the two real-time RT-PCR assays, direct conventional RT-PCR assay, and virus isolation assay on human rhabdomyosarcoma cells (RD cells). The total positive rate of both HEV71 and CVA16 was 69.48% with real-time RT-PCR assay, 47.42% with RT-PCR assay, and 34.58% with virus isolation assay. One HFMD clinical specimen was positive for HEV, but negative for HEV71 or CVA16, which was identified as Echovirus 11 (Echo11) by virus isolation, RT-PCR, and sequencing for the VP1 gene. The two real-time RT-PCR assays had been applied in 31 provincial HFMD labs to detect the pathogens of HFMD, which has contributed to the rapid identification of the pathogens in the early stages of HFMD outbreaks, and helped to clarify the etiologic agents of HFMD in China.     

A novel multiplex PCR assay for Salmonella subspecies identification

Aim:  To develop a novel multiplex polymerase chain reaction (PCR) assay with six primer pairs for Salmonella subspecies identification.
Methods and Results:  Five primer pairs were chosen to detect the genes ( fljB , mdcA , gatD , stn and STM4057) responsible for several phenotypic traits or encoding (sub) species-specific regions. A primer pair for invA was added to simultaneously detect Salmonella . The combination of these primer pairs was expected to give unique results to all subspecies, including Salmonella bongori. The multiplex PCR assay was optimized and evaluated with 53 Salmonella strains representing all S. enterica subspecies, S. bongori and five non- Salmonella strains. The multiplex PCR assay revealed that the genotypes were well correlated with the phenotypes in the Salmonella strains tested. The unique band patterns to their subspecies were generated from 94·3% (50/53) of the Salmonella strains, and no product from other strains by the multiplex PCR assay.
Conclusions:  The multiplex PCR assay we developed was found to be a rapid, specific and easy to perform method compared with traditional biochemical tests for Salmonella subspecies identification, especially for rapid screening of large numbers of samples.
Significance and Impact of the Study:  The assay will be useful for characterizing Salmonella isolates from reptiles, which belong to various subspecies, and therefore add to the scientific understanding of reptile-associated Salmonellosis.     

Sharing habitat: Effects of migratory barnacle geese density on meadow breeding waders

• 1.Following targeted conservation actions most goose populations have increased. The growing goose populations caused an increase in human-wildlife conflicts and have the potential to affect nature values. As meadow birds, including meadow-breeding waders, were declining throughout Western Europe, the possible negative effect of rising numbers of foraging barnacle geese on their breeding success has been questioned.
• 2.We used GPS-transmitter data to measure the density of foraging barnacle geese during daylight hours. Using dynamic Brownian Bridge Movement Models (dBBMM), we investigated the effect of barnacle goose density on the territory distribution of five wader species, and on nest success of the locally common Northern lapwing. We used model selection methods to identify the importance of barnacle goose density related to other environmental factors.
• 3.Our results showed an insignificant positive association between barnacle goose density and nest territory density of the Northern lapwing and common redshank. Barnacle goose density had no influence on territory selection of godwit, oystercatcher and ringed plover. We did, however, find a negative correlation between barnacle geese density and the nest success of the Northern Lapwing.
• 4.We infer that either barnacle goose foraging leads to improved territory conditions for some wader species, or that both barnacle geese and waders prefer the same type of habitat for foraging and nesting. Higher barnacle goose density was correlated with fewer Northern lapwing nests being successful.
• 5.Synthesis and application: Experimental research is needed to disentangle the causal chain, but based on our observational findings, we suggest to increase water logging that may attract both barnacle geese and wader species. Further investigation on the effects of barnacle geese on wader species is necessary to identify the cause of the negative correlation between barnacle geese density and nest success of lapwings; nest protection experiments could give further insight.

     

Breeding barnacle geese in Kolokolkova Bay,Russia: number of breeding pairs,reproductive success and morphology

We report the results of an expedition to a barnacle-goose (Branta leucopsis) breeding area in Kolokolkova Bay, west of the lower Pechora delta in northern Russia, undertaken in July 2002. In total, 6 breeding colonies were found within the study area, harbouring 1,324 nests. Mean clutch size was 2.77±0.10 but may have been underestimated because of nest predation. Nest predation was high and correlated with the density of breeding gulls, Larus. The 2002 season was relatively cold and peak hatch occurred late, on 14 July. More than 11,000 barnacle geese were found to moult in the area which, together with the large number of nests found, emphasises the importance of Kolokolkova Bay for barnacle geese. Adult barnacle geese (341) were captured, marked and measured during their annual wing moult. Birds with broods started to moult approximately 2 weeks later than non- and failed breeders. Weight loss during moult was 3 times as rapid as reported for barnacle geese breeding in the Baltic, and a large cost of reproduction seemed to exist in the form of reduced body weight at the onset of moult for birds leading broods. Work in the area will continue over the coming years to document and explain the differences in major life-history parameters, dynamics and environmental effects between arctic and temperate breeding barnacle-goose populations.     

A novel simple one-step single-tube RT-duplex PCR method with an internal control for detection of bovine viral diarrhoea virus in bulk milk, blood, and follicular fluid samples.

A simple one-step single-tube RT-PCR method was developed for detection of bovine viral diarrhea virus (BVDV) in bulk milk, blood and follicular fluid samples. A set of universal primers (UTR DL1/4) was designed for RT-PCR to detect BVDV type I and II viruses simultaneously and was tested for efficacy in comparison to published primers for two type strains, 42 field isolates, and 95 diagnostic samples. The sensitivity (100%) and specificity (96.2%) of the RT-PCR assay, with the universal primers for 95 diagnostic samples, were equal to those of virus isolation. An internal control targeting a bovine actin gene was also included in the same reaction tube to monitor RNA preparation and RT-PCR procedure. A total of 632 specimens (378 bulk milk, 140 blood, and 112 follicular samples) were tested in the year 2000 by the RT-duplex PCR assay in parallel with virus isolation. The one-step single-tube RT-duplex PCR with the universal primers UTR DL1/4 was sensitive, specific, less complicated and cost effective for detection of BVDV in various types of specimens.     

Development of one-step TaqMan(R) real-time reverse transcription-PCR and conventional reverse transcription PCR assays for the detection of equine rhinitis A and B viruses

ABSTRACT: BACKGROUND: Equine rhinitis viruses A and B (ERAV and ERBV) are common equine respiratory viruses belonging to the family Picornaviridae. Sero-surveillance studies have shown that these two viral infections are prevalent in many countries. Currently, the diagnosis of ERAV and ERBV infections in horses is mainly based on virus isolation (VI). However, the sensitivity of VI testing varies between laboratories due to inefficient viral growth in cell culture and lack of cytopathic effect. Therefore, the objective of this study was to develop molecular diagnostic assays (real-time RT-PCR [rRT-PCR] and conventional RT-PCR [cRT-PCR] assays) to detect and distinguish ERAV from ERBV without the inherent problems traditionally associated with laboratory diagnosis of these infections. RESULTS: Three rRT-PCR assays targeting the 5'-UTR of ERAV and ERBV were developed. One assay was specific for ERAV, with the two remaining assays specific for ERBV. Additionally, six cRT-PCR assays targeting the 5'-UTR and 3D polymerase regions of ERAV and ERBV were developed. Both rRT-PCR and cRT-PCR assays were evaluated using RNA extracted from 21 archived tissue culture fluid (TCF) samples previously confirmed to be positive for ERAV (n = 11) or ERBV (n = 10) with mono-specific rabbit antisera. The ERAV rRT-PCR and cRT-PCR assays could only detect ERAV isolates and not ERBV isolates. Similarly, the ERBV rRT-PCR and cRT-PCR assays could only detect ERBV isolates and not ERAV isolates. None of the rRT-PCR or cRT-PCR assays cross-reacted with any of the other common equine respiratory viruses. With the exception of one cRT-PCR assay, the detection limit of all of these assays was 1 plaque forming unit per ml (pfu/ml). CONCLUSION: The newly developed rRT-PCR and cRT-PCR assays provide improved diagnostic capability for the detection and differentiation of ERAV and ERBV. However, a larger number of clinical specimens will need to be tested before each assay is adequately validated for the detection of ERAV and/or ERBV in suspect cases of either viral infection.     

Detection of Aphanomyces astaci in North American crayfish by polymerase chain reaction

We present a PCR based method to detect Aphanomyces astaci in North American crayfish. Primers were designed to specifically amplify parts of the internal transcribed spacer (ITS) regions and the 5.8 rRNA gene of A. astaci. A single round and a semi-nested assay were tested for their sensitivity and specificity. Specificity of the PCR assays was tested against several closely related Aphanomyces species, other Oomycetes and some non-A. astaci DNA that might be found in or on crayfish. The single round assay was fully specific against all DNA tested. In the semi-nested assay, cross-reaction was seen when the equivalent of 40,000 or more genomic units of A. invadans or A. frigidophilus were entered into the PCR reaction. The lower detection limit of both assays lies around 1 genomic unit of A. astaci. Investigation of various parts of the exoskeleton of 3 North American crayfish species revealed that for O. limosus and P. leniusculus the telson and soft abdominal cuticle yielded a positive PCR reaction most frequently. For the third species, Procambarus clarkii, only 1 individual tested positive, so no conclusion as to preferred infestation site(s) could be drawn.     

Detection and differentiation of Cryptosporidium by real-time polymerase chain reaction in stool samples from patients in Rio de Janeiro, Brazil

This study reports the first genetic characterisation of Cryptosporidium isolates in Brazil using real-time polymerase chain reaction (RT-PCR). A total of 1,197 faecal specimens from children and 10 specimens from human immunodeficiency virus-infected patients were collected between 1999-2010 and screened using microscopy. Forty-eight Cryptosporidium oocyst-positive isolates were identified and analysed using a generic TaqMan assay targeting the 18S rRNA to detect Cryptosporidium species and two other TaqMan assays to identify Cryptosporidium hominis and Cryptosporidium parvum. The 18S rRNA assay detected Cryptosporidium species in all 48 of the stool specimens. The C. parvum TaqMan assay correctly identified five/48 stool samples, while 37/48 stool specimens were correctly amplified in the C. hominis TaqMan assay. The results obtained in this study support previous findings showing that C. hominis infections are more prevalent than C. parvum infections in Brazil and they demonstrate that the TaqMan RT-PCR procedure is a simple, fast and valuable tool for the detection and differentiation of Cryptosporidium species.     

Detection of tumor mutations in the presence of excess amounts of normal DNA

Mutations are important markers in the early detection of cancer. Clinical specimens such as bodily fluid samples often contain a small percentage of mutated cells in a large background of normal cells. Thus, assays to detect mutations leading to cancer need to be highly sensitive and specific. In addition, they should be possible to carry out in an automated and high-throughput manner to allow large-scale screening. Here we describe a screening method, termed PPEM (PNA-directed PCR, primer extension, MALDI-TOF), that addresses these needs more effectively than do existing methods. DNA samples are first amplified using peptide nucleic acid (PNA)-directed PCR clamping reactions in which mutated DNA is preferentially enriched. The PCR-amplified DNA fragments are then sequenced through primer extension to generate diagnostic products. Finally, mutations are identified using matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This method can detect as few as 3 copies of mutant alleles in the presence of a 10,000-fold excess of normal alleles in a robust and specific manner. In addition, the method can be adapted for simultaneous detection of multiple mutations and is amenable to high-throughput automation.     

Patch choice of avian herbivores along a migration trajectory–From Temperate to Arctic

Migratory waterfowl species seem to track temporal and spatial pulses of optimal forage availability on their way from temperate wintering to arctic breeding sites. In order to unravel the relative contribution of forage quality and forage biomass to foraging choices in avian herbivores, we experimentally manipulated biomass and quality of main forage plants through fertilisation and grazing exclusion at three sites along the flyway of barnacle geese, Branta leucopsis. Fertilisation increased the nitrogen content of the forage and grazing exclusion increased biomass levels. Manipulated plots were offered to wild geese in a random block experimental design and goose visitation was measured through dropping counts. At all sites there was a trend towards a higher preference of plots with increased quality and average biomass above plots with an average quality and increased biomass. Generally, geese preferred plots with highest standing crop of nitrogen. The numerical response of the geese to forage changes was supported by behavioural observations at the Baltic site. We conclude that for migrating barnacle geese the bottlenecks in the standing crop of nitrogen appear to lie in the limited biomass availability at the Baltic stopover site and the limited nutrient content of food in the Arctic breeding site, restricting the potential nutrient intake on these sites.