泌尿生殖道沙眼衣原体omp1基因多态性研究

从中国不同城市收集疑为沙眼衣原体(Ct)感染的泌尿生殖道标本323份,巢式PCR扩增Ctomp1基因片段(包括4个变异区),测定其中96份阳性标本omp1基因序列,根据同源性分型并分析其多态位点;根据氨基酸序列,用Mega 3软件构建进化树,分析临床株与相应参考株之间的亲缘关系。从96份沙眼衣原体阳性标本中,检出28种基因变体,其中E型最常见;同时发现Ct E、F型omp1基因高度保守,而其它基因型都显示一定的变异性。进化树分析发现,各临床株与相应参考株之间遗传距离较近。实验结果表明沙眼衣原体omp1基因呈现较大的多态性,可为其疫苗的研制及感染的防治提供重要的实验依据。     

Chlamydia trachomatis serovar distribution and other sexually transmitted coinfections in subjects attending an STD outpatients clinic in Italy

We studied the prevalence of Chlamydia trachomatis (CT) urogenital infection and the distribution of different genotypes in a non-selected STD population of 1625 patients, evaluating presence of coinfections with other sexually transmitted diseases. Each patient was bled to perform serological tests for syphilis and HIV, then urethral or endocervical swabs were obtained for the detection of CT and Neisseria gonorrhoeae by culture. DNA extracted from remnant positive swabs was amplified by omp1 Nested PCR and products were sequenced. Total prevalence of CT infection was 6.3% (103/1625), with strong differences between men and women (11.4% vs 3.9%, P<0.01). Clinical symptoms and coinfections were much more frequent in men than in women (P<0.01). The most common serovar was E (prevalence of 38.8%), followed by G (23.3%), F (13.5%) D/Da (11.6%) and J (4.8%). Serovars distribution was statistically different between men and women (P=0.042) and among patients with or without coinfection (P=0.035); patients infected by serovar D/Da showed the highest coinfection rate. This study can be considered a contribution in increasing knowledge on CT serovar distribution in Italy. Further studies are needed to better define molecular epidemiology of CT infection and to investigate its correlation with other STDs.     

Lateral gene transfer of streptococcal ICE element RD2 (region of difference 2) encoding secreted proteins

Background  

The genome of serotype M28 group A Streptococcus (GAS) strain MGAS6180 contains a novel genetic element named Region of Difference 2 (RD2) that encodes seven putative secreted extracellular proteins. RD2 is present in all serotype M28 strains and strains of several other GAS serotypes associated with female urogenital infections. We show here that the GAS RD2 element is present in strain MGAS6180 both as an integrative chromosomal form and a circular extrachromosomal element. RD2-like regions were identified in publicly available genome sequences of strains representing three of the five major group B streptococcal serotypes causing human disease. Ten RD2-encoded proteins have significant similarity to proteins involved in conjugative transfer of Streptococcus thermophilus integrative chromosomal elements (ICEs).     

Structures and genetics of Kdo-containing O-antigens of Cronobacter sakazakii G2706 and G2704, the reference strains of serotypes O5 and O6

Cronobacter sakazakii G2706 and G2704 are the reference strains of serotypes O5 and O6 in the serological classification of this species proposed recently. Mild acid degradation of the lipopolysaccharides of both strains resulted in cleavage of the O-polysaccharide chains at the acid-labile linkage of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) to yield oligosaccharides representing repeating units of the O-polysaccharides. The oligosaccharides and alkali-degraded lipopolysaccharides were studied by sugar analysis along with 1D and 2D ¹H and ¹³C NMR spectroscopy, and the following O-polysaccharide structures were established:The structure of strain G2706 is unique among the known bacterial polysaccharide structures, whereas that of strain G2704 is identical to the structure of Cronobacter malonaticus 3267 [MacLean, L. L.; Vinogradov, E.; Pagotto, F.; Farber, J. M.; Perry, M. B. Biochem. Cell Biol.2009, 87, 927–932], except for that the latter lacks O-acetylation. Putative functions of the genes in the O-antigen gene clusters of C. sakazakii strains studied are in agreement with the O-polysaccharide structures.     

Population-based genetic and evolutionary analysis of Chlamydia trachomatis urogenital strain variation in the United States

Chlamydia trachomatis is a major cause of ocular and sexually transmitted diseases worldwide. While much of our knowledge about its genetic diversity comes from serotyping or ompA genotyping, no quantitative assessment of genetic diversity within serotypes has been performed. To accomplish this, 507 urogenital samples from a multicenter U.S. study were analyzed by phylogenetic and statistical modeling. No B, Da, or I serotypes were represented. Based on our analyses, all but one previous urogenital B serotype was identified as Ba. This, coupled with the lack of B serotypes in our population, suggests that B has specific tropism for ocular mucosa. We identified a Ba/D recombinant (putative crossover nucleotide 477; P < 0.0001) similar to a B/D mosaic we described previously from an African trachoma patient. Computational analyses of the Ba/D recombinant indicated that upstream changes were less important for tissue tropism than downstream incorporation of the D sequence. Since most serotypes had nonsynonymous/synonymous ratios of <1.0, the major outer membrane protein, encoded by ompA, has many functional constraints and is under purifying selection. Surprisingly, all serotype groups except for J had a unimodal population structure indicating rapid clonal expansion. Of the groups with a unimodal structure, E and Ia and, to a lesser extent, G and K were prevalent, had infrequent incorporation of mutations, and, compared to other groups, had a relatively greater degree of diversifying selection, consistent with a selective sweep of mutations within these groups. Collectively, these data suggest a diverse evolutionary strategy for different serogroups of the organism.     

Development and characterization of a potent producer of penicillin G amidase by mutagenization

Summary The penicillin G amidase (PGA) activity of a parent strain of E. coli (PCSIR-102) was enhanced by chemical mutagenization with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). After screening and optimization, a penicillinase deficient mutant (MNNG-37) was isolated and found effective for the production of penicillin G amidase as compared to the parent strain of E. coli (PCSIR-102). Penicillin G amidase activity of MNNG-37 appeared during an early stage of growth, whereas PCSIR-102 did not exhibit PGA activity due to the presence of penicillinase enzyme which inhibits the activity of enzyme PGA. However, MNNG-37 gave a three-fold increase in enzyme activity (231 IU mg⁻¹) as compared to PCSIR-102 (77 IU mg⁻¹) in medium containing 0.15 and 0.1% concentrations of phenylacetic acid, respectively which was added after 6 h of cultivation. The difference in K _m values of the enzyme produced by parent strain PCSIR-102 (0.26 mM) and mutant strain MNNG-37 (0.20 mM) is significant (1.3-fold increase in K _m value) which may show the superiority of the latter in terms of better enzyme properties.     

Reproductive Physiology in Young Men Is Cumulatively Affected by FSH-Action Modulating Genetic Variants: FSHR -29G/A and c.2039 A/G,FSHB -211G/T

Follicle-Stimulating Hormone Receptor (FSHR) -29G/A polymorphism (rs1394205) was reported to modulate gene expression and reproductive parameters in women, but data in men is limited. We aimed to bring evidence to the effect of FSHR -29G/A variants in men. In Baltic young male cohort (n = 982; Estonians, Latvians, Lithuanians; aged 20.2±2.0 years), the FSHR -29 A-allele was significantly associated with higher serum FSH (linear regression: effect 0.27 IU/L; P = 0.0019, resistant to Bonferroni correction for multiple testing) and showed a non-significant trend for association with higher LH (0.19 IU/L) and total testosterone (0.93 nmol/L), but reduced Inhibin B (−7.84 pg/mL) and total testes volume (effect −1.00 mL). Next, we extended the study and tested the effect of FSHR gene haplotypes determined by the allelic combination of FSHR -29G/A and a well-studied variant c.2039 A/G (Asn680Ser, exon 10). Among the FSHR -29A/2039G haplotype carriers (A-Ser; haplotype-based linear regression), this genetic effect was enhanced for FSH (effect 0.40 IU/L), Inhibin B (−16.57 pg/mL) and total testes volume (−2.34 mL). Finally, we estimated the total contribution of three known FSH-action modulating SNPs (FSHB -211G/T; FSHR -29G/A, c.2039 A/G) to phenotypic variance in reproductive parameters among young men. The major FSH-action modulating SNPs explained together 2.3%, 1.4%, 1.0 and 1.1% of the measured variance in serum FSH, Inhibin B, testosterone and total testes volume, respectively. In contrast to the young male cohort, neither FSHR -29G/A nor FSHR haplotypes appeared to systematically modulate the reproductive physiology of oligozoospermic idiopathic infertile patients (n = 641, Estonians; aged 31.5±6.0 years). In summary, this is the first study showing the significant effect of FSHR -29G/A on male serum FSH level. To account for the genetic effect of known common polymorphisms modulating FSH-action, we suggest haplotype-based analysis of FSHR SNPs (FSHR -29G/A, c.2039 A/G) in combination with FSHB -211G/T testing.     

Serotypes and mating types of clinical strains ofCryptococcus neoformans isolated in Taiwan

Twenty-one strains ofCryptococcus neoformans isolated from patients in Taiwan were characterized for serotypes and mating types. Slide agglutination test was performed with 8 factor-specific sera (Iatron Company, Japan) to determine the serotypes. Wheat bran agar (WBA) and malt extract agar (MEA, Wickerham) media were used for the mating tests. Twenty of the isolates were of serotype A, and one was serotype B. Except for 2 strains of serotype A, all of the serotype A strains mated withFilobasidiella neoformans var.neoformans, mating type a. The only serotype B strain mated withF. neoformans var.bacillispora mating type a in MEA medium. These data revealed the low prevalence (1/21; 4.8%) ofC. neoformans var.gattii in Taiwan, a subtropically located island.     

Comparison of OspA Serotypes for Borrelia burgdorferi Sensu Lato from Japan,Europe and North America

Sixty-one Borrelia burgdorferi sensu lato strains from various sources (ticks, human, and wild animals) in Japan and two strains from ticks in Far Eastern Russia were classified on the basis of reactivity with 16 monoclonal antibodies (mAb) to outer surface protein A (OspA) and by DNA-DNA hybridization assay. Eleven OspA serotypes (J1 to J11) were recognized among the Japanese and the Far East Russian isolates (serotypes J1 to J9 were identified as B. garinii, serotype J10 was identified as B. afzelii, and serotype J11 corresponded to B. japonica), whereas 7 OspA serotypes for North American and European isolates previously reported (Bettina Wilske et al, J. Clin. Microbiol. 31:340-350, 1993) were not observed except for OspA serotype 2 which showed identical reactivity with OspA serotype J10. This finding provides helpful information for understanding the geographical distribution of Lyme disease borrelia and the development of vaccine and diagnostic tests. In conclusion: 1. B. burgdorferi sensu stricto has not been observed in Japan, 2. Japanese B. afzelii isolates are closely related to those from Europe, 3. B. garinii isolates from Japan are highly heterogeneous and apparently different from European B. garinii isolates.     

Prevalence of Zoonotic Giardia duodenalis Assemblage B and First Identification of Assemblage E in Rabbit Fecal Samples Isolates from Central China

Giardia duodenalis is an important zoonotic pathogen, causes diarrhea in humans and animals worldwide. To date, few data are available on the prevalence of G. duodenalis in rabbits in China. In total, 955 fecal samples were collected from rabbits during 2008–2011 in Henan Province, Central China. The overall prevalence of G. duodenalis was 8.4% (80/955) on microscopic analysis, with the highest infection rate (11.3%) in rabbits aged 91–200 d. All G. duodenalis‐positive isolates were characterized at the small subunit ribosomal RNA, β‐giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase genes. Two assemblages and a mixed assemblage were detected in the rabbits: assemblage B (n = 26), assemblage E (n = 2), and a mixed assemblage of B and E (n = 4). Assemblage B isolates showed variability at the nucleotide sequences belonging to the so‐called subtype BIV, based on analysis of multiple genes. This is the first report of G. duodenalis assemblage E in rabbits, and one novel subtype of assemblage E was identified through sequence analysis of gdh and bg genes, respectively. Our data suggest that rabbits may be reservoirs of G. duodenalis cysts potentially infectious to humans.     

Improved production of erythromycin A by expression of a heterologous gene encoding S-adenosylmethionine synthetase

An S-adenosylmethionine synthetase (SAM-s) gene from Streptomyces spectabilis was integrated along with vector DNA into the chromosome of a Saccharopolyspora erythraea E2. Elevated production of SAM was observed in the recombinant strain Saccharopolyspora erythraea E1. The results from the bioassay showed that the titer of erythromycin was increased from 920 IU ml⁻¹ by E₂ to approximately 2,000 IU ml⁻¹ by E₁. High performance liquid chromatography (HPLC) analysis revealed that there was a 132% increase in erythromycin A compared with the original strain, while the erythromycin B, the main impurity component in erythromycin, was decreased by 30%. The sporulation process was inhibited, while the SAM-s gene was expressed. The addition of the exogenous SAM also inhibited sporulation and promoted an increase in erythromycin titers. An erratum to this article can be found at     

Production in <Emphasis Type="Italic">Trichoderma reesei</Emphasis> of three xylanases from <Emphasis Type="Italic">Chaetomium thermophilum</Emphasis>: a recombinant thermoxylanase for biobleaching of kraft pulp

Three endoxylanase genes were cloned from the thermophilic fungus Chaetomium thermophilum CBS 730.95. All genes contained the typical consensus sequence of family 11 glycoside hydrolases. Genomic copies of Ct xyn11A, Ct xyn11B, and Ct xyn11C were expressed in the filamentous fungus T. reesei under the control of the strong T. reesei cel7A (cellobiohydrolase 1, cbh1) promoter. The molecular masses of the Ct Xyn11A, Ct Xyn11B, and Ct Xyn11C proteins on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were 27, 23, and 22 kDa, respectively. Ct Xyn11A was produced almost as efficiently as the homologous xylanase II from a corresponding single-copy transformant strain. Ct Xyn11B production level was approximately half of that of Ct Xyn11A. The amount of Ct Xyn11C was remarkably lower. Ct Xyn11A had the highest temperature optimum and stability of the recombinant xylanases and the highest activity at acid-neutral pH (pH 5–7). It was the most suitable for industrial bleaching of kraft pulp at high temperature.     

Optimization of Lipase Production from <Emphasis Type="Italic">Aspergillus terreus</Emphasis> by Response Surface Methodology and Its Potential for Synthesis of Partial Glycerides Under Solvent Free Conditions

Aspergillus terreus produces lipase 7.01 IU/ml in 96 h after optimization by one variable at a time method. Using the significant factors i.e. corn oil (A), sodium nitrate (B), casein (C), agitation rate (D) and incubation period (E) RSM was carried out resulting in 19.65 IU/ml from the combination +1(A), −1(B), −1(C), +1(D) and 0(E). The interactions between sodium nitrate, casein and agitation with corn oil were most significant. Scale up of production from 250 ml shake flask to 30 l bioreactor resulted in increased productivity of 0.52 IU/ml/h as against 0.2 IU/ml/h obtained in shake flasks. This lipase could carryout solvent free synthesis of partial glycerides of oleic acid with 96% efficiency in 12 h.     

鸡源大肠杆菌毒力基因检测及分子流行特征

[目的] 本试验旨在阐明鸡源大肠杆菌致病性及分子流行特性,为探索大肠杆菌流行途径制定合理的防控策略提供新思路。[方法] 2018–2019年在河北省采集病死鸡肝脏样品,通过选择培养基筛选、生化鉴定、血清凝集试验对分离菌株进行系统鉴定,应用PCR方法检测分离株中毒力基因流行情况。参考系统发育群分类方法对大肠杆菌进行分群分析,并参照McMLST网站数据库提供的7对管家基因序列进行多位点序列分型（multilocus sequence typing,MLST）分析。[结果] 结果显示,56株分离株符合大肠杆菌生化特征,分为8个生化表型,B4（30.36%）、B5（25%）和B2（23.21%）为主要生化表型。56株分离株大肠杆菌血清凝集试验均呈阳性,分为11种血清型,O₇₈（26.79%）、O₂（23.21%）、O₁₅₇（17.86%）和O₁（14.29%）为主要流行血清型。56株大肠杆菌共检测出15种肠外大肠杆菌毒力基因,未检出papC、ibeA和ibeB基因。黏附相关基因fimC和抗血清存活因子相关基因ompA携带率为100%。aatA、yijP、irp2、mat、iss,检出率分别为98.21%、98.21%、98.21%、96.43%、92.86%。同时,大肠杆菌与铁转运相关基因iroN、fyuA、iucD、irp2检出率均在80%以上。56株大肠杆菌中有20株属于肠出血型大肠杆菌（enterohemorrhagic E.coli,EHEC）,其次是肠聚集型大肠杆菌（enteroaggregative E.coli,EAEC）（n=4）、肠产毒素型大肠杆菌（Enterotoxigenic E.coli,ETEC）（n=2）。这些菌株D群分离株较多,其次是B2群。通过MLST分型分析,共分为22个ST型,其中ST88（n=7）、ST85（n=6）、ST243（n=6）型为主要流行型。[结论] 结果显示大肠杆菌血清型多样,毒力因子种类繁多,致病性大肠杆菌同时携带多种毒力基因,表明动物源大肠杆菌具有较强的毒力基础。     

Characterization of the functional activity of botulinum neurotoxin subtype B6

Clostridium botulinum produces the highly potent neurotoxin, botulinum neurotoxin (BoNT), which is classified into seven serotypes (A–G); the subtype classification is confirmed by the diversity of amino acid sequences among the serotypes. BoNT from the Osaka05 strain is associated with type B infant botulism and has been classified as BoNT/B subtype B6 (BoNT/B6) by phylogenetic analysis and the antigenicity of its C‐terminal heavy chain (H_C) domain. However, the molecular bases for its properties, including its potency, are poorly understood. In this study, BoNT/B6 holotoxin was purified and the biological activity and receptor binding activity of BoNT/B6 compared with those of the previously‐characterized BoNT/B1 and BoNT/B2 subtypes. The derivative BoNT/B6 was found to be already nicked and in an activated form, indicating that endogenous protease production may be higher in this strain than in the other two strains. BoNT/B1 exhibited the greatest lethal activity in mice, followed by BoNT/B6, which is consistent with the sensitivity of PC12 cells. No significant differences were seen in the enzymatic activities of the BoNT/Bs against their substrate. H_C/B1 and H_C/B6 exhibited similar binding affinities to synaptotagmin II (SytII), which is a specific protein receptor for BoNT/B. Binding to the SytII/ganglioside complex is functionally related to the toxic action; however, the receptor recognition sites are conserved. These results suggest that the distinct characteristics and differences in biological sensitivity of BoNT/B6 may be attributable to the function of its H_c.domain.

     

Enterocytozoon bieneusi,Giardia, and Cryptosporidium Infecting White‐tailed Deer

Despite a white‐tailed deer (WTD) population in the United States of approximately 32 million animals extremely little is known of the prevalence and species of the protists that infect these animals. This study was undertaken to determine the presence of potential human protist pathogens in culled WTD in central Maryland. Feces from fawns to adults were examined by molecular methods. The prevalence of Enterocytozoon bieneusi, Cryptosporidium, and Giardia was determined by PCR. All PCR‐positive specimens were sequenced to determine the species and genotype(s). Of specimens from 80 WTD, 26 (32.5%) contained 17 genotypes of E. bieneusi. Four genotypes were previously reported (I, J, WL4, LW1) and 13 novel genotypes were identified and named DeerEb1‐DeerEb13. Genotypes I, J, and LW1 are known to infect humans. Ten (12.5%) specimens contained the Cryptosporidium deer genotype, and one (1.25%) contained Giardia duodenalis Assemblage A. The identification zoonotic G. duodenalis Assemblage A as well as four E. bieneusi genotypes previously identified in humans suggest that WTD could play a role in the transmission of those parasites to humans.     

Whole Genome Sequencing Based Characterization of Extensively Drug-Resistant Mycobacterium tuberculosis Isolates from Pakistan

Improved molecular diagnostic methods for detection drug resistance in Mycobacterium tuberculosis (MTB) strains are required. Resistance to first- and second- line anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, these SNPs can vary between MTB lineages therefore local data is required to describe different strain populations. We used whole genome sequencing (WGS) to characterize 37 extensively drug-resistant (XDR) MTB isolates from Pakistan and investigated 40 genes associated with drug resistance. Rifampicin resistance was attributable to SNPs in the rpoB hot-spot region. Isoniazid resistance was most commonly associated with the katG codon 315 (92%) mutation followed by inhA S94A (8%) however, one strain did not have SNPs in katG, inhA or oxyR-ahpC. All strains were pyrazimamide resistant but only 43% had pncA SNPs. Ethambutol resistant strains predominantly had embB codon 306 (62%) mutations, but additional SNPs at embB codons 406, 378 and 328 were also present. Fluoroquinolone resistance was associated with gyrA 91–94 codons in 81% of strains; four strains had only gyrB mutations, while others did not have SNPs in either gyrA or gyrB. Streptomycin resistant strains had mutations in ribosomal RNA genes; rpsL codon 43 (42%); rrs 500 region (16%), and gidB (34%) while six strains did not have mutations in any of these genes. Amikacin/kanamycin/capreomycin resistance was associated with SNPs in rrs at nt1401 (78%) and nt1484 (3%), except in seven (19%) strains. We estimate that if only the common hot-spot region targets of current commercial assays were used, the concordance between phenotypic and genotypic testing for these XDR strains would vary between rifampicin (100%), isoniazid (92%), flouroquinolones (81%), aminoglycoside (78%) and ethambutol (62%); while pncA sequencing would provide genotypic resistance in less than half the isolates. This work highlights the importance of expanded targets for drug resistance detection in MTB isolates.     

CYP1B1 (4326C > G), CYP2F1 (c.14_15insC), CYP2J2 (?76G > T), and CYP2S1 (13106C > T and 13255A > G) polymorphisms and genetic predisposition to chronic respiratory diseases induced by smoking and occupational factors

The contribution of the polymorphic markers of cytochrome P450 genes to respiratory diseases caused by smoking and occupational factors has been assessed. For this purpose, PCR-RFLP analysis of the CYP1B1 (rs1056836, 4326C > G), CYP2F1 (rs11399890, c.14_15insC), CYP2J2 (rs890293, -76G > T), and CYP2S1 (rs34971233, 13106C > T and rs338583, 13255A > G) gene polymorphisms has been performed. The analysis has shown that CYP1B1 (rs1056836, 4326C > G) and CYP2F1 (rs11399890, c.14_15insC) polymorphisms may contribute to the development of occupational chronic bronchitis. The proportion of CYP1B1*1*3 heterozygotes in the group of patients with occupational chronic bronchitis is considerably greater than in the group of healthy workers (69.16% versus 53.29%; χ² = 5.94, p = 0.02, p _cur = 0.04, OR = 1.97, the 95% CI is 1.13–3.42). Patients with occupational chronic bronchitis and healthy workers significantly differed from each other in the CYP2F1 genotypes frequency distribution (rs11399890, c.14_15insC) (χ² = 6.18, d.f. = 2, p = 0.05). CYP2F1 wild type/ins heterozygous genotype frequency is higher in healthy workers (36.08%) than in patients (22.22%) (χ² = 5.48, p = 0.02, p _cur = 0.04, OR = 0.51, the 95% CI is 0.28–0.90). No association has been found between the CYP2J2 (rs890293, −76G > T) or CYP2S1 (rs34971233, 13106C > T, and rs338583, 13255A > G) gene polymorphisms and respiratory diseases.     

NBS1基因SNPs与中国汉族人群原发性肝癌的遗传易感性

为分析DNA损伤修复相关基因NBS1单核苷酸多态性(SNPs)与原发性肝癌遗传易感性的关系,并对高分辨率单链构象多态性(SSCP)检测技术在SNPs分型中的适用性进行评估,本研究对来自中国汉族人群的327例原发性肝癌以及295例阴性对照中NBS1基因常见SNPs的稀有等位基因频率进行检测和分析．此外,对NBS1基因6个常见SNPs分别选择部分样本同时进行直接序列测定,以比较2种方法的检测效果．119例原发性肝癌以及95例肝硬化/慢性肝炎组织标本的SSCP分析结果表明,6个常见NBS1基因SNPs位点(102G>A, 320+208G/A, 553G>C, 1197T>C, 2016A>G和2071-30A>T)中,SNP 1197T>C的稀有等位基因频率为68.1%,显著高于肝硬化/慢性肝炎对照的57.9% (P = 0.0298)．对该SNP位点另外采用208份肝细胞癌和200份健康人群血液标本进一步分析, 肝细胞癌SNP 1197T>C的稀有等位基因频率为66.8%,显著高于健康人群对照的58.8% (P = 0.0170)．其他5个SNPs的稀有等位基因频率在原发性肝癌与肝硬化/慢性肝炎之间均无显著性差异．高分辨率SSCP分析法与直接序列测定法对所选样本的SNPs基因分型结果完全一致,而且直接测序法对PCR扩增产物质量的要求相对高分辨率SSCP分析更高．研究表明,中国汉族人群NBS1基因SNP 1197T>C可能与原发性肝癌的发生相关,高分辨率SSCP技术准确度与直接测序法相当,且操作更加简便易行,非常适用于大量样本多个已知SNPs的基因分型．     

Identification of surface protective antigen (spa) types in Erysipelothrix reference strains and diagnostic samples by spa multiplex real‐time and conventional PCR assays

Aim: To develop spa multiplex real‐time and conventional PCR assays to detect and differentiate between spaA, spaB and spaC genes within Erysipelothrix spp. Methods and Results: For evaluation of the assays, 28 Erysipelothrix spp. reference strains, 25 tissues from pigs inoculated with reference strains of serotypes 1, 2, 5, 10 or 18, and 15 diagnostic samples were used. SpaA was found to be present in Erysipelothrix rhusiopathiae serotypes 1a, 1b, 2, 5, 9, 12, 15, 16, 17, 23 and N; spaB was detected in E. rhusiopathiae serotypes 4, 6, 8, 11, 19 and 21 and spaC was detected in E. sp. strain 2 serotype 18. Spa‐related genes were not detected in E. tonsillarum strains (serotypes 3, 7, 10, 14, 20, 22, 24, 25, 26) or E. sp. strain 1 (serotype 13). With the spa multiplex real‐time PCR assay, it was also possible to further differentiate spaB into spaB1 (serotypes 4, 6, 8, 19 and 21) and spaB2 (serotype 11). Overall, spaA was detected in seven experimental tissue samples and six diagnostic tissue samples, and spaC in two experimental tissue samples. The detection limits were determined to be five colony‐forming units (CFU) per reaction for the spa multiplex real‐time PCR assay and 4000 CFU per reaction for the conventional PCR assay. Conclusions: Both spa PCR assays were specific and reproducible in the identification of spa types in Erysipelothrix spp. Significance and Impact of the Study: The described spa PCR assays may be useful tools for investigating spa prevalence among strains isolated from field tissues and to determine the role of the Spa proteins in vaccine protection and pathogenesis.