A new outbreak of fox rabies at the Russian–Mongolian border

<正>Dear Editor,Lake Baikal and its neighboring territories are an intermediate zone for the"steppe"and"arctic-like"rabies virus lineages in Russia.After the elimination of dog-mediated rabies during the early 1980s,this area remained rabies-free for over 25–30 years.A sudden reappearance of rabies occurred in this zone in the Republic of Buryatia in 2011–2012.A marginal part of the Mongolian steppe penetrates the Siberian taiga forests in this area,and human and animal rabies have been repeatedly recorded in the Republic of Buryatia from the end of the     

Investigation and analysis of rabies viral infection and distribution in China in 2005–2012

Dear Editor, We report the results of a preliminary investigation of data collected between 2005 and 2012.A National Human Rabies Surveillance program was initiated in high rabies incidence regions in 2005,and subsequently carried out nationally to monitor rabies situation.Our work presents a summary of epidemiological and etiologic rabies surveillance data collected since the implementation of the program.     

SYBR Green I-based product-enhanced reverse transcriptase assay for quantification of retroviral PFV and detection of the divalent cation preference of PFV RT

<正>Dear Editor,Prototype foamy virus(PFV)belongs to the genus Spumavirus in the Spumaretrovirinae subfamily of Retroviridae.Although PFV and HIV have much in common,research into PFV has lagged far behind that into HIV,as PFV appeared to be non-pathogenic both in accidentally infected humans and in experimentally infected animals.In recent decades,however,more attention has been focused on PFV because it seems to be     

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.     

A real-time PCR-based quantitative assay for 3-methylcytosine demethylase activity of ALKBH3

Human AlkB homolog 3 (ALKBH3), a homolog of the Escherichia coli protein AlkB, demethylates 1-methyladenine and 3-methylcytosine (3-meC) in single-stranded DNA and RNA by oxidative demethylation. Immunohistochemical analyses on clinical cancer specimens and knockdown experiments using RNA interference in vitro and in vivo indicate that ALKBH3 is a promising molecular target for the treatment of prostate, pancreatic, and non-small cell lung cancer. Therefore, an inhibitor for ALKBH3 demethylase is expected to be a first-in-class molecular-targeted drug for cancer treatment. Here, we report the development of a novel, quantitative real-time PCR-based assay for ALKBH3 demethylase activity against 3-meC by highly active recombinant ALKBH3 protein using a silkworm expression system. This assay enables us to screen for inhibitors of ALKBH3 demethylase, which may result in the development of a novel molecular-targeted drug for cancer therapy.     

A quantitative real-time polymerase chain reaction assay for the seagrass pathogen Labyrinthula zosterae

The protist Labyrinthula zosterae (Phylum Bigyra, sensu Tsui et al. 2009) has been identified as a causative agent of wasting disease in eelgrass (Zostera marina), of which the most intense outbreak led to the destruction of 90% of eelgrass beds in eastern North America and western Europe in the 1930s. Outbreaks still occur today, albeit at a smaller scale. Traditionally, L. zosterae has been quantified by measuring the necrotic area of Z. marina leaf tissue. This indirect method can however only lead to a very rough estimate of pathogen load. Here, we present a quantitative real-time polymerase chain reaction (qPCR) approach to directly detect and quantify L. zosterae in eelgrass tissue. Based on the internal transcribed spacer (ITS) sequences of rRNA genes, species-specific primers were designed. Using our qPCR, we were able to quantify accurately and specifically L. zosterae load both from culture and eelgrass leaves using material from Europe and North America. Our detection limit was less than one L. zosterae cell. Our results demonstrate the potential of this qPCR assay to provide rapid, accurate and sensitive molecular identification and quantification of L. zosterae. In view of declining seagrass populations worldwide, this method will provide a valuable tool for seagrass ecologists and conservation projects.     

A rapid quantitative real-time PCR-based DNA quantification assay coupled with species--assignment capabilities for two hybridizing Macaca species

Regional populations of rhesus and long-tailed macaques exhibit fundamental differences in mitochondrial DNA, short tandem repeat and single nucleotide polymorphism variation between mainland and insular Southeast Asian populations. Some studies have revealed genetic admixture between these species due to natural hybridization and human-assisted intercrosses. A quantitative real-time PCR (qPCR) assay was developed to efficiently determine the species of origin of a macaque biological sample, and to quantify the species-specific template DNA. Prior knowledge of species identity and DNA concentrations are crucial for maintaining cost-effective methods and accurate DNA analysis. DNA from 109 regionally representative rhesus and long-tailed macaques was qPCR amplified to determine the species and template quantities. Of the 19 Vietnamese long-tailed macaques, 3 samples were discovered to be hybrids.     

抗—HBs国际单位RIA一点法定量测定与计算方法的探讨

在Radio-immuno Assay(RIA)试验测抗－HBs国际单位的简易定量与计算中,采用RIA一点法测定,以分析表达式mIU/ml＝SC（mIU/ml)[exp(0.69315S-NC/SC-NC-1]进行结果计算。此式可以常用对数式表示为mIU/ml=SC(mIU/ml)[lg^-1(0.30103S-NC/SC-NC-1],当（S－NC）在0．3－1．1界线内时,其计算结果的相对误差小于6％,是目前误差最小的简易计算方法。同时还推算求出Holliger公式的阳性对照的最适含量为124．26mIU／ml,Richardson公式的阳性对照为125mIU/ml,中国药品生物制品检定所公式的阳性对照为92．5mIU/ml。     

A nested real-time PCR assay has an increased sensitivity suitable for detection of viruses in aerosol studies

Aims:  Influenza is commonly spread by infectious aerosols; however, detection of viruses in aerosols is not sensitive enough to confirm the characteristics of virus aerosols. The aim of this study was to develop an assay for respiratory viruses sufficiently sensitive to be used in epidemiological studies.
Method:  A two-step, nested real-time PCR assay was developed for MS2 bacteriophage, and for influenza A and B, parainfluenza 1 and human respiratory syncytial virus. Outer primer pairs were designed to nest each existing real-time PCR assay. The sensitivities of the nested real-time PCR assays were compared to those of existing real-time PCR assays. Both assays were applied in an aerosol study to compare their detection limits in air samples.
Conclusions:  The nested real-time PCR assays were found to be several logs more sensitive than the real-time PCR assays, with lower levels of virus detected at lower Ct values. The nested real-time PCR assay successfully detected MS2 in air samples, whereas the real-time assay did not.
Significance and Impact of the Study:  The sensitive assays for respiratory viruses will permit further research using air samples from naturally generated virus aerosols. This will inform current knowledge regarding the risks associated with the spread of viruses through aerosol transmission.     

大鼠尿激酶SYBR Green I real time RT-PCR引物的设计

设计用于SYBR Green I法实时定量逆转录多聚酶链反应(QRT-PCR)检测大鼠尿激酶型纤溶酶原激活因子(uPA)mRNA的引物。从基因库获取靶基因及相关序列,充分收集争分析相关生物信息学数据,应用Oligo 6.22设计出一对长度为21bp的引物,其GG含量为52.4%;上下游引物3’最稳定二聚体和及发夹结构的能量分别为-1.5、-0.40 kcal/mol和-3.5、-O.90 kcal/mol,引物间最稳定二聚体为-3.1 kcal/mol。5’端和中间△G值较高,高于3’端△G;引发效率分别455和403。实验证明,该引物能够高效、特异地实现对靶序列的检测,适用于SYBR Green I法实时定量检测(uPA)mRNA。     

A partial genome assay for quantitative trait loci in wheat (Triticum aestivum) using different analytical techniques

F₁ plants between two intervarietal chromosome substitution lines of European spring wheat varieties, Sicco (Chinese Spring 5B) and Highbury (Chinese Spring 5B), were used to produce 114 doubled haploid lines, 45 by the Hordeum bulbosum technique and 69 by anther culture. These two sets of lines were characterized for variation at a range of morphological, isozyme and RFLP marker loci, and genetic maps were developed with emphasis on chromosomes 6B, 7A, 7B and 7D. A subset of lines, scored for production traits in field trials in 1986 and 1987, were analysed for quantitative trait loci (QTL). The performance of the lines for the quantitative traits studied showed no overall differences due to the method of production of the lines. QTL were located on the linkage map for ear emergence time, height, tiller weight, yield and 50-grain weight using four analytical methods. Many of these effects showed genotype x year interaction.     

A multi-dose serological assay suitable to quantify the potency of inactivated rabies vaccines for veterinary use

The mouse vaccination-challenge test, which is the most widely used method for determining the potency of inactivated rabies vaccines, is imprecise, time-consuming, and causes severe distress to the test animals. An alternative single-dose serological method has been implemented in the European Pharmacopoeia Monograph 0451 to replace the mouse challenge test for batch release. This single-dose limit method provides semi-quantitative results, but is not suitable for quantifying potency. We have now extended this serological method to a multi-dose format which allows a quantification of vaccine potency. In studies including all rabies vaccine strains relevant for Europe, we found dose-dependency for all vaccines and standard preparations. We have demonstrated that the multi-dose serological approach provides reliable quantitative potency results and is more precise than the mouse vaccination-challenge test. We have shown that adjuvanted vaccines can be calibrated against non-adjuvanted material, and that reference material can be calibrated against the International Standard. The method is therefore capable of assigning potency with the additional advantage of requiring fewer animals and reducing distress. Once the applicability of the method has been further verified in a collaborative study, it can complement the single-dose assay and eventually eliminate the need for the mouse challenge test.     

A quantitative real-time PCR assay for the highly sensitive and specific detection of human faecal influence in spring water from a large alpine catchment area

AIMS: The aim of the study was the development of a sensitive human-specific quantitative real-time PCR assay for microbial faecal source tracking (MST) in alpine spring water. The assay detects human-specific faecal DNA markers (BacH) from 16S rRNA gene sequences from the phylum Bacteroidetes using TaqMan minor groove binder probes. METHODS AND RESULTS: The qualitative and quantitative detection limits of the PCR assay were 6 and 30 marker copies, respectively. Specificity was proved by testing 41 human faeces and waste water samples and excluding cross-amplification from 302 animal faecal samples from Eastern Austria. Marker concentrations in human faecal material were in the range from 6.6 x 10(9) to 9.1 x 10(10) marker equivalents per gram. The method was sensitive enough to detect a few 100 pg of faeces in faecal suspensions. The assay was applied on water samples from an alpine karstic spring catchment area and the results reflected the expected levels of human faecal influence. CONCLUSIONS: The method exhibited sufficient sensitivity to allow quantitative source tracking of human faecal impact in the investigated karstic spring water. Significance AND IMPACT OF THE STUDY: The developed method constitutes the first quantitative human-specific MST tool sensitive enough for investigations in ground and spring water.     

Quadruplex real-time quantitative PCR assay for the detection of pathogens related to late-onset ventilator-associated pneumonia: A preliminary report

A quadruplex real-time (RT) qPCR assay for the detection and quantification in 4 h of Staphylococcusaureus, Pseudomonasaeruginosa, Acinetobacterbaumannii and Stenotrophomonasmaltophilia directly from bronchoalveolar lavage specimens was developed. The specificity of the assay was 100% for all four species.