Triplex real‐time polymerase chain reaction assay for detection and quantification of norovirus (GI and GII) and sapovirus

To improve detection of norovirus (NoVGI, NoVGII) and sapovirus (SaV), a simultaneous quantitative RT‐PCR method was established. This triplex real‐time PCR method was evaluated using a combination of optimized specific primers and probes. The performance of the developed PCR assay was equivalent to that of monoplex real‐time PCR across a broad dynamic range of 10²–10⁷ copies/assay using plasmid DNA standards. The limit of detection was 10² copies/assay. The quantitative value was comparable with that of monoplex real‐time PCR of stool samples. Our triplex real‐time PCR is useful for detection of NoV and SaV infections.     

Mitochondrial genome of the Komodo dragon: efficient sequencing method with reptile-oriented primers and novel gene rearrangements.

The mitochondrial genome of the Komodo dragon (Varanus komodoensis) was nearly completely sequenced, except for two highly repetitive noncoding regions. An efficient sequencing method for squamate mitochondrial genomes was established by combining the long polymerase chain reaction (PCR) technology and a set of reptile-oriented primers designed for nested PCR amplifications. It was found that the mitochondrial genome had novel gene arrangements in which genes from NADH dehydrogenase subunit 6 to proline tRNA were extensively shuffled with duplicate control regions. These control regions had 99% sequence similarity over 700 bp. Although snake mitochondrial genomes are also known to possess duplicate control regions with nearly identical sequences, the location of the second control region suggested independent occurrence of the duplication on lineages leading to snakes and the Komodo dragon. Another feature of the mitochondrial genome of the Komodo dragon was the considerable number of tandem repeats, including sequences with a strong secondary structure, as a possible site for the slipped-strand mispairing in replication. These observations are consistent with hypotheses that tandem duplications via the slipped-strand mispairing may induce mitochondrial gene rearrangements and may serve to maintain similar copies of the control region.     

一种简便、快捷的胰蛋白酶抑制剂基因的分离与克隆方法

从 3个豇豆品种幼嫩叶片中分离出核基因组 DNA,参照已知的几种 Bowman-Birk型胰蛋白酶抑制剂基因序列 ,设计合成了 2 7bp,且含有 Bam H I位点的寡核苷酸引物 ,分别以 3种豇豆核基因组 DNA为模板 ,PCR扩增 ,均得到长度约为 3 40 bp的 DNA片段。产物 DNA片段经 DNA序列分析 ,结果表明三者的碱基序列相同 ,与报道的胰蛋白酶抑制剂基因相比 ,同源性为 1 0 0 %和 99.7%。     

Expression of a novel tropomyosin isoform in axolotl heart and skeletal muscle

TPM1κ is an alternatively spliced isoform of the TPM1 gene whose specific role in cardiac development and disease is yet to be elucidated. Although mRNA studies have shown TPM1κ expression in axolotl heart and skeletal muscle, it has not been quantified. Also the presence of TPM1κ protein in axolotl heart and skeletal muscle has not been demonstrated. In this study, we quantified TPM1κ mRNA expression in axolotl heart and skeletal muscle. Using a newly developed TPM1κ specific antibody, we demonstrated the expression and incorporation of TPM1κ protein in myofibrils of axolotl heart and skeletal muscle. The results support the potential role of TPM1κ in myofibrillogenesis and sarcomeric function. J. Cell. Biochem. 110: 875–881, 2010. © 2010 Wiley‐Liss, Inc.     

Complete coding sequence and molecular analysis of hepatitis A virus from a chimpanzee with fulminant hepatitis

Background Hepatitis A virus (HAV) infects both humans and non‐human primates, in experimentally infected chimpanzees is typically milder than in humans. In 1982, Abe and Shikata reported a first case of a chimpanzee with fulminant hepatitis caused by spontaneous HAV infection, and the underlying mechanisms of the disease remain unknown. Methods To characterize denoted CFH‐HAV, we conducted cloning and near full‐length sequence analysis. Results Phylogenetic analyses of VP1‐2A and complete sequence comparison between various genotypes and the sample sequence showed clustering in genotype IB. Based on BLAST analysis, the sequence was most closely related to the wild‐type (HM175/WT) isolate. Amino acid and nucleic acid similarities were 99.8% and 94.41%, respectively. Conclusions The chimpanzee may have been infected with human HAV genotype IB. The substitutions in VP2, VP4, 2B, 2C, and 3D, which may enhance virus proliferation, contributed to disease severity culminating in fulminant hepatic failure.     

实验性变态反应性脑脊髓炎大鼠发病中脑组织血红素氧合酶-1基因和蛋白表达的动态变化

为探讨脑组织血红素氧合酶-1(hemeoxygenase-1,HO-1)对实验性变态反应性脑脊髓炎(experimental allergic encephalomyelitis,EAE)的作用,分别应用逆转录-聚合酶链反应(RT-PCR)和免疫组化技术测定了豚鼠脊髓生理盐水匀浆 完全福氏佐剂诱导EAE大鼠1、7、14、21d时,脑组织HO-1基因和蛋白表达的动态变化,并观察与症状之间的关系。结果显示：对照组大鼠脑组织仅有少量HO-1基因和蛋白表达：诱导EAE后,伴随着大鼠EAE症状及脑组织病理损伤的出现和进行性加重,脑组织HO-1基因和蛋白表达量逐渐增高。在豚鼠脊髓生理盐水匀浆 完全福氏佐剂诱导7d后,HO-1 mRNA上升至高峰。14d时,HO-1蛋白表达至高峰,HO-1阳性细胞主要位于脉络丛、穹隆下器、血管“套袖样”病灶的周围,与EAE病变部位一致。此时大鼠的病情最重、体重减轻最显著、脑组织病理改变最明显。21d时脑组织HO-1基因和蛋白表达量逐渐下降,大鼠EAE症状也逐渐减轻。应用HO-1特异性抑制剂锡原卟啉-9以抑制脑内HO-1蛋白表达后,大鼠EAE症状和脑组织损伤明显减轻,说明脑组织HO-1的动态变化与EAE症状及脑组织损伤密切相关。提示脑组织HO-1基因和蛋白过表达对EAE发病起着重要的作用,应用HO-1抑制剂可能是防治该病的有效方法之一。     

Name that microbe: rapid identification of taxa responsible for individual fragments in fingerprints of microbial community structure

Polymerase chain reaction (PCR)‐based ‘fingerprinting’ methods, such as Terminal restriction fragment length polymorphism, Length Heterogeneity‐Polymerase Chain Reaction (LH‐PCR) and Automated Ribosomal Intergenic Spacer Analysis (ARISA) make possible quantitative studies of microbial community structure and dynamics. Here we outline a strategy for the rapid and cost‐effective isolation of 16S clones corresponding to particular fragment sizes in a fingerprint, based on applying the fingerprinting method to pools of colonies from a clone library. This allows the definitive identification of taxa responsible for the most important bands in the community fingerprint from a full 16S sequence. It offers significant advantages over random selection of clones and removes a significant barrier to the use of these methods.     

Increased reliability of selective PCR by using additionally mutated primers and a commercialTaq DNA polymerase enhancer

A reliable selective PCR procedure that combines the use of additionally mutated primers with the specificity-enhancing properties of a commercial preparation (Perfect Match, Stratagene) is described. The human immunodeficiency virus type 1pol gene point mutations known to confer in vitro resistance to azidothymidine were examined as a model for optimization of the assay. The usual strategy of deliberately introducing an additional mismatch 1 residue from the 3′ end in the wild-type and mutant primers did not allow reproducible discrimination between wild-type and mutant target sequences. Addition of minimal amounts of Perfect Match to the same PCR mixtures resulted in a significantly enlarged range of selective annealing temperatures, providing a valuable and cost-effective means for reliable detection of known mutations by selectivePCR.     

A novel mutation in the myeloperoxidase gene in a Chinese female with complete myeloperoxidase deficiency: The role of nonsense-mediated mRNA decay

Myeloperoxidase (MPO) is an important enzyme in innate immunity. Here, we describe the first identified Chinese individual with complete MPO deficiency. The proband was ascertained through routine automated complete blood analysis. Analysis of MPO function and immunogenicity revealed that MPO levels in neutrophils were significantly decreased. Mutational analysis revealed a novel premature termination codon p.(Trp602*) in exon 11 of the MPO gene, which was inherited in an autosomal recessive manner. We demonstrated that nonsense-mediated mRNA decay is involved in the molecular pathology of MPO deficiency in this case. The study of MPO deficiency can be helpful in understanding the function and biosynthesis mechanisms of MPO.     

Genetic analysis of ORF5 in porcine reproductive and respiratory syndrome virus in Japan

In recent years, no reports regarding genetic information on porcine reproductive and respiratory syndrome virus (PRRSV) with a focus on Japan have been published. To clarify the effect of time on PRRSV genomic evolution, we sequenced the open reading frame 5 (600 or 603 bases) obtained from Japanese PRRSV isolates for three periods (1992-1993, 2000-2001, and 2007-2008) and compared their phylogenetic relationships. Assessment of mean pairwise homology of nucleotide sequences of PRRSV isolates indicated a trend towards increasing heterogeneity over time. In addition, we newly detected a virus classified in cluster IV, indicative of the increasing genetic variation of PRRSV in Japan.