Genotyping of 11 human papillomaviruses by multiplex PCR with a GeXP analyzer

A new, rapid, and high-throughput method was developed for simultaneous detection of 11 human papillomavirus (HPV) genotypes including nine high-risk types (HPV16, 18, 31, 33, 35, 39, 52, 58, and 66) and two low-risk types (HPV6 and 11) in a single tube by multiplex PCR based on a GenomeLab Gene Expression Profiler (GeXP) analyzer (GeXP-PCR). Eleven sets of chimeric primers were used to initiate the PCR, and one pair of universal primers was used for the subsequent cycles of the PCR. The specificity of GeXP-PCR for each HPV type was examined with clinical samples of single type HPV infection tested previously. The sensitivity of GeXP-PCR was evaluated by performing the assay on serial 10-fold dilutions of cloned PCR products. The GeXP-PCR achieved a sensitivity of 100 copies when all of the 11 pre-mixed plasmids containing HPV targets were present. Analyses of 124 clinical specimens using the GeXP-PCR demonstrated that the GeXP-PCR assay had comparable sensitivity and specificity to those of reported multiple PCR assay and an increased detection of HPV 11 in samples with mixed infections. In conclusion, the GeXP-PCR is a fast, sensitive, and high throughput method for the detection of multiple HPV infections.     

Molecular characterization of norovirus GII strains identified in Albania

Noroviruses (NoVs) are considered as the leading cause of diarrheal diseases in all groups of age. In the last decade the number of NoV outbreaks worldwide is increasing. Data published by the systems of NoV surveillance show the GII.4 strain as the human predominant genotype circulating worldwide and new genetic variants of GII.4 were associated with epidemic events. In Albania the economy transformation has damaged significantly the environment and a large circulation of enteric viruses was reported in the past with the presence of NoV among the genotyped strains. This study aimed to characterize, by molecular analysis, the NoV GII strains detected in Albania during two time periods: in 2010 from the outbreak occurred in Ballsh and in 2002–2003 from sporadic cases of diarrhoea. A total of 21 Nov GII strains were characterized. The NoV GII.4 was genotyped more frequently and it was related closely to the pandemic variants recorded in GenBank. During 2002–2003, six NoV GII recombinant strains have been characterized. J. Med. Virol. 85:731–736, 2013. © 2013 Wiley Periodicals, Inc.     

Variant of hepatitis B virus isolated in zimbabwe

The polymerase chain reaction (PCR) was used to amplify an approximately 1.2 kb DNA fragment encompassing the pre-S/S gene region of HBV DNA from serum of patients with acute hepatitis B virus infection. Nucleotide sequence analysis revealed a number of interesting features in the S gene region. Two Bam HI sites were located at nucleotide positions 557 and 872, respectively, in the S gene. Guanine (G) was found at nucleotide position 903 as part of AGA, the codon for arginine (R) corresponding to amino acid position 122 of the S protein. Ade-nine (A) was found at nucleotide position 1017 as part of AXIA, the codon for lysine (K) corresponding to amino acid position 160 of the S protein. Nucleotide sequence alignment revealed a 97% homology to the corresponding domain of an HBVadw genome (clone pFDW294). Within the second loop of the “a” determinant, two mutations resulting in substitution of serine or threonine with the hydrophobic amino acids, methio-nine at position 143 and with alanine in place of glycine at position 145, are predicted from the consensus nucleotide sequence of the PCR-derived clones. Subtyping with monoclonal antibodies showed that the HBsAg was of the ayw subtype. © 1994 Wiiey-Liss, Inc.     

Rapid preparation of diagnostic probes for the fragile X syndrome by direct PCR amplification of human chromosomal DNA

Summary The fragile X syndrome is a common familial form of mental retardation and is associated with a rare fragile site at Xq27.3 (FRAXA). This disorder has recently been reported to correlate with length variations of restriction genomic DNA fragments which may due to the amplification of (CCG)_n trinucleotide repeats located at the FRAXA locus. We described here a rapid preparation method of diagnostic DNA probes for the fragile X syndrome by direct enzymatic amplification of human chromosomal DNA. ThePstI-assay, which is Southern blot analysis of DNA samples probed by PCR products, was shown to be sensitive method for diagnostic purposes to detect the size variations specific in the fragile X syndrome.     

HLA-DRB1 genotyping by modified PCR-RFLP method combined with group-specific primers

We previously introduced HLA-DQA1, -DPB1 and DQB1 genotyping with the modified PCR-RFLP method using some informative restriction enzymes which have either a single cleavage site or alternatively no cleavage site in the amplified DNA region, depending on the HLA alleles, making reading of RFLP band patterns much easier. In this study, 43 HLA-DRB1 alleles, excluding DRB1*1103 and *1104 for which no restriction enzymes are available to distinguish each from the other, could be defined by this modified PCR-RFLP method combined with 7 pairs of group-specific primers. It is impossible to distinguish DRB1*0701 and DRB1*0702 as they are identical for the second exon of DRB1. For DR1-DRB1, DR2-DRB1, DR4-DRB1, DR7-DR1, DR9-DRB1, DRw10-DRB1 or DRw52 associated antigens (DR3, w11, w12, w13, w14, and DRw8)-DRB1 gene amplification, the second exon of the DRB1 gene was selectively amplified using each group-specific primer from genomic DNAs of 70 HLA-homozygous B-cell lines and healthy Japanese by PCR. Amplified DNAs were digested with restriction endonucleases and then subjected to electrophoresis assaying simply for cutting, or no cutting, of the DNA, although some alleles can be distinguished only after examination of RFLP band patterns generated and in some cases using double digestion technique with two restriction enzymes. This modified PCR-RFLP method can be successfully applied to all possible DRB1 heterozygotes, despite the fact that 15 pairs of heterozygotes among them cannot be distinguished theoretically by the PCR-SSO method, because the PCR-RFLP method can tell whether two polymorphic sites are linked to each other (cis position) or located on a different chromosome (trans position) by checking the length of RFLP bands generated with double digestion. Thus, the PCR-RFLP method is technically simple, practical and inexpensive for determination of the HLA-DRB1 alleles for routine HLA typing work.     

Genotype distribution of hepatitis C virus infection in Taiwan

To investigate the prevalence of genotype distribution of hepatitis C virus (HCV) infection in Taiwan, genotypes were identified in 122 (36 anti-HCV-positive blood donors, 44 anti-HCV-positive aborigines, 28 hemodialysis patients, and 14 patients with chronic liver diseases) of 280 subjects, using polymerase chain reaction by Okamoto's type-specific primer method. Type II was the dominant (66.7%) type among anti-HCV-positive blood donors, followed by type III and type IV with the same percentages (16.7%), while none of type I was detected. The prevalence of genotype distribution were 75.0%, 81.1%, and 64.3% for type 11,4.6%, 17.9%, and 21.4% for type III, 13.6%, 0%, and 7.1% for type IV, for the aborigines, hemodialysis, and chronic liver diseases groups, respectively. Four subjects revealed mixed infections by two different genotypes: two cases of II and III; and each one case of II and IV, and III and IV. Diverse genotype distributions in two hemodialysis groups disclose the existence of obvious regional differences even within a region. The results reveal the highest prevalence of type II as in Japan. However, there is a higher prevalence rate of type IV than in Japan. © 1994 Wiley-Liss, inc.     

Diagnostic assays developed for the control of foot-andmouth disease in India

Foot-and-mouth disease(FMD) is a highly contagious and economically devastating disease of livestock, primarily affecting cattle, buffalo and pigs. FMD virus serotypes O, A and Asia1 are prevalent in India and systematic efforts are on to control and eventually eradicate the disease from the country. FMD epidemiology is complex due to factors like co-circulation, extinction, emergence and re-emergence of genotypes/lineages within the three serotypes, animal movement, diverse farm practices and large number of susceptible livestock in the country. Systematic vaccination, prompt diagnosis, strict biosecurity measures, and regular monitoring of vaccinal immunity and surveillance of virus circulation are indispensible features for the effective implementation of the control measures. Availability of suitable companion diagnostic tests is very important in this endeavour. In this review, the diagnostic assays developed and validated in India and their contribution in FMD control programme is presented.     

A nested polymerase chain reaction for detection of Legionella pneumophila in clinical specimens

Objective: Because presently used methods for diagnosis of Legionella pneumonia lack sufficient sensitivity and sometimes specificity and rapidity, the detection of Legionella spp. by amplification of nucleic acids might be valuable. However, performing polymerase chain reaction (PCR) on clinical samples such as sputum is difficult because of the presence of extraneous DNA and inhibitors of the reaction. An attempt to circumvent these problems was made.
Method: A nested PCR method was devised using primers from the mip gene of Legionella pneumophila. This PCR was tested on pure cultures of legionellae and clinical isolates of other bacteria. Clinical samples (bronchoalveolar lavage fluid, bronchial aspirate and sputum) from patients who suffered from legionellosis and samples from patients who suffered from other causes of pneumonia were also tested.
Results: The PCR was specific for L. pneumophila and no non- Legionella bacteria reacted. Ten to 50 colony forming units of Legionella in the sample could be detected. Twenty-two of 25 clinical samples were positive among patients suffering from pneumonia proven to be due to L. pneumophila serogroups 1, 3, 4, 5 and 6. Two of the three negative samples were from patients who had been treated with adequate therapy for at least 2 days and were culture negative. However, nine other culture-negative samples were PCR positive, of which seven came from patients who had been treated for 3–7 days. All pneumonia patients in the control group proved negative in PCR. A commercial kit for DNA preparation from clinical samples, based on absorption of nucleic acids to silica gel, was superior to the traditional phenol/chloroform extraction and increased the rapidity, simplicity and sensitivity of the procedure.
Conclusions: A nested, simplified and rapid PCR method using mip primers proved to be more sensitive than culture and as sensitive and specific as other PCR procedures previously reported.     

Detection of multiple viral and bacterial infections in acute exacerbation of chronic obstructive pulmonary disease: A pilot prospective study

Few studies have evaluated the contribution of multiple virus and bacterial infections in acute exacerbation of chronic obstructive pulmonary disease. This study estimated the burden of multiple viral and bacterial respiratory infections in moderate to very severe chronic obstructive pulmonary disease patients that were prospectively followed‐up during a 12‐month pilot study. Clinical data were collected monthly and sputum was collected at the time of each acute exacerbation event. Classical culture techniques for bacteria and multiplex polymerase chain reaction (PCR) and microarray detection assays were performed to identify viral and atypical bacterial pathogens in the sputum. Overall, 51 patients were included and 45 acute exacerbation events were investigated clinically and microbiologically. Among the 45 acute exacerbation events, 44% had evidence of viral infection involving human rhinovirus (HRV) and metapneumovirus (hMPV) in 20% and 18%, respectively. Intracellular bacteria were not found in sputum by PCR. Common bacterial pathogens were identified in 42% of acute exacerbation patients, most frequently Branhamella catarrhalis, Streptococcus pneumoniae and Haemophilus influenzae. Viral or virus and bacteria co‐infections were detected in 27% of acute exacerbation events (n = 12) with HRV and hMPV involved in 92% of cases. Patients with co‐infections did not present greater clinical severity scores at exacerbation and more recurrence of acute exacerbation events at 3 and 6 months than those with single infections (P > 0.4). These results suggest that HRV and hMPV may be contributors or cofactors of AECOPD. These findings indicate that viral or virus and bacterial co‐infections do not impact significantly on the clinical severity of acute exacerbation of chronic obstructive pulmonary disease and recurrence at 3 and 6 months. J. Med. Virol. 85:866–873, 2013. © 2013 Wiley Periodicals, Inc.     

在石蜡切片中进行微切割-PCR-银染的方法

目的：探索在石蜡切片中应用微切割－ＰＣＲ－银染技术,检测大肠癌的微卫星不稳定性和杂合性缺失。方法：用微切割技术在２８例石蜡切片中提取淋巴细胞和间质细胞、癌旁粘膜腺管、不典型地生腺管、腺瘤腺管和腺癌细胞,每例至少包含３种类型细胞,经蛋白酶Ｋ消化后,直接用于ＰＣＲ扩增,产物进行８％变异聚丙烯酰胺凝胶电泳－银染。共进行了ＴＧＦ－βＲⅡ（Ａ）１０、ｈＭＳＨ２（Ａ）２６－Ｂａｔ２６,ｈＭＳＨ３（Ａ）８,ｈＭＳＨ６（Ｃ）８４个微卫星位点的检测。结果：ｈＭＳＨ３（Ａ）８和ｈＭＳＨ６（Ｃ）８位点在２８例标本全部扩增出目的片段,ＴＧＦ－βＲⅡ（Ａ）１０和ｈＭＳＨ２（Ａ）２６位点则分别有２３例、２２例扩增出目的片段,且ｈＭＳＨ２（Ａ）２６位点有５例癌细胞呈阳性,其中１例同时有腺癌细胞阳性。结论：微切割－ＰＣＲ－银染技术应用于石蜡切     

Clonality analysis of defined B-cell populations in archival tissue sections using microdissection and the polymerase chain reaction

A simple microdissection technique involving the use of a drawn-out glass pipette was developed for isolation of defined cell subsets from tissue sections. Using this technique and the polymerase chain reaction (PCR), clonally rearranged immunoglobulin (Ig) heavy chain genes were reliably amplified in single neoplastic follicles or few hundreds of tumour cells isolated from archival haematoxylin and eosin or immunostained sections of B-cell lymphomas. A polyclonal nature was consistently demonstrated in reactive lymphoid follicles or interfollicular reactive B-cells within the same lymphoma sections. Microdissection of lymphoma cells from within foci of chronic inflammation improved the resolution of tumour-specific PCR products by reducing amplification of background polyclonal B-cell sequences. The combination of microdissection and PCR techniques, therefore, provides an important tool for the investigation of B-cell lymphomas and also allows simple and specific access for other molecular genetic analyses of different cell subsets on tissue sections.     

Detection of human herpesvirus 8 DNA sequences in peripheral blood mononuclear cells of children

Human herpesvirus 8 (HHV-8) DNA sequences were examined in peripheral blood mononuclear cell (PBMC) DNA samples of 56 children, 15 healthy adults, and 10 renal transplant patients by the polymerase chain reaction (PCR). The PCR amplification was carried out using the published KS330₂₃₃ primer pairs to amplify HHV-8 DNA sequences. The PCR-amplified products were confirmed by Southern blot hybridization with radiolabeled 233 bp HHV-8 DNA fragment, which was cloned and sequenced from the PCR-amplified product of Kaposi's sarcoma tissue. Six PCR-amplified product of four children and two renal transplant patients were cloned and sequenced. HHV-8 DNA sequences were detected in 36 of 56 (64%) normal children, in 12 of 15 (80%) healthy adults, and in all 10 renal transplant patients by Southern blot hybridization of PCR-amplified products. Six PCR-amplified products were confirmed by sequencing. These results suggest that HHV-8 infection is prevalent in the Japanese population with infection occurring in early childhood. J. Med. Virol. 53:81–84, 1997. © 1997 Wiley-Liss, Inc.     

Loop-mediated isothermal amplification method for detection of human papillomavirus type 6, 11, 16, and 18

A new method was developed for detection of human papillomavirus (HPV) by loop-mediated isothermal amplification (LAMP), which was compared with the polymerase chain reaction (PCR), and real-time PCR for specificity and sensitivity. All initial validation studies with the control DNA proved to be type-specific. In order to evaluate the reliability of HPV type-specific LAMP detecting HPV DNA from clinical samples, tissue specimens were obtained from 27 patients with external genital polypoid lesions. The histologic diagnoses included condyloma acuminatum (n = 21), bowenoid papulosis (n = 2), seborrheic keratosis (n = 2), epidermolytic acanthoma (n = 1), and hairy nymphae (n = 1). HPV-6 DNA and HPV-11 DNA were detected in 18 and 3 of 21 condylomata acuminata, respectively, and there was no simultaneous infection. HPV-16 DNA was detected in one of two bowenoid papuloses. HPV DNA was not detected in the seborrheic keratoses, epidermolytic acanthoma, and hairy nymphae. These results correlated perfectly with those from real-time PCR analysis. Most positive samples contained high copy numbers of HPV DNA. HPV-11 DNA was detected in one case that could not be detected by PCR. The average reaction time was about 59 min. There was a linear correlation between the genome quantity and reaction time to reach the threshold. The LAMP method has an additional advantage as a quantitative method, and is superior in terms of sensitivity, specificity, rapidity, and simplicity, and can potentially be a valuable tool for the detection of HPV DNA.     

Investigation of specimen mislabelling in paraffin-embedded tissue using a rapid, allele-specific, PCR-based HLA class II typing method

Mislabelling of surgical specimens can seriously affect the accuracy of histopathology reports. We describe two cases in which clinically suspected mislabelling was investigated by polymerase chain reaction (PCR)-based HLA DRB and DQB tissue typing of paraffin biopsy-derived DNA, using sequence specific primers (PCR-SSP HLA typing). In the first case, two patients underwent surgery for breast carcinoma. A subcutaneous lymph node containing metastatic carcinoma was received with the breast specimen from one patient, but was clinically considered more likely to originate from the other patient who underwent breast surgery on the same day. In the second case, histological examination of retained products of conception from a young woman revealed adenocarcinoma, but a repeat curettage specimen consisted of secretory phase endometrium. In case 1, PCR-SSP HLA typing confirmed the clinical suspicion that the subcutaneous lymph node received with tissue from one patient originated from the other patient. This result converted the first patient from lymph node positive breast carcinoma to lymph node negative disease. In case 2, there was no evidence from PCR-SSP HLA typing that the endometrial samples had originated from different patients. PCR-SSP HLA typing requires fewer steps than methods based on PCR amplification followed by oligonucleotide probing (PCR-SSOP HLA typing), and relies on the amplification of shorter sequences of DNA. Therefore, this technique can produce more rapid results than PCR-SSOP HLA typing, and is ideally suited to typing partially degraded DNA derived from formalin-fixed and paraffin-embedded tissue.     

冠状动脉粥样硬化性心脏病患者载脂蛋白(a)五核苷酸重复序列基因多态性的初步研究

目的　探讨载脂蛋白 (a) [apolipoprotein(a) ,apo(a) ]五核苷酸重复序列 (pentanucleotiderepeats,PNR)基因多态性与中国汉族人冠状动脉粥样硬化性心脏病 (简称冠心病 )发病的关系。方法　应用聚合酶链反应结合聚丙烯酰胺凝胶电泳和硝酸银染色 ,对 15 3名中国汉族正常人和 16 5例冠心病患者apo(a) PNR基因多态性进行了分析。结果　冠心病组 apo(a) PNR(TTTTA) 5/8基因型频率 (0 .188)和(TTTTA) 5等位基因频率 (0 .115 )显著高于正常对照组 (0 .0 39,0 .0 2 6 ) ,差异有显著性 (P <0 .0 1,P<0 .0 5 )。结论　apo(a) PNR基因多态性与人群易患冠心病有关 ,可能在一定程度上参与了冠心病的发生和发展过程。     

Asp57-negative HLA DQ beta chain and DQA1*0501 allele are essential for the onset of DQw2-positive and DQw2-negative coeliac disease.

The genetic predisposition to coeliac disease is associated with the HLA DQw2 allele. Coeliac patients lacking the DQw2 allele are very rare and always exhibit the DR4-DQw3 haplotype. We performed oligotyping of polymerase chain reaction (PCR)-amplified DQA1 and DQB1 genes in six DQw2-negative and 30 DQw2-positive coeliac patients. The DQB analysis showed that all six DQw2-negative patients possessed the DQB1*0302 allele. The other DQB alleles found in five of these patients were DQB1*0501, DQB1*0604 and DQB1*0302. The DQ beta chains encoded from all these alleles have the replacement of aspartic acid residue at position 57 (Asp57), as well as the DQB1*0201 allele which was found in all 30 DQw2-positive coeliac patients. The DQw2-negative proband who lacked the homozygous Asp57 replacement exhibited the DQA1*0501 allele in the DQA1 gene. The DQA1*0501 allele was also found in 27 of the 30 DQw2-positive coeliac patients. Among this group of coeliacs, the four cases lacking the DQA1*0501 allele exhibited the homozygous Asp57 replacement in the DQ beta chain. Our results indicate that Asp57-negative DQ beta alleles are involved in both DQw2-positive and -negative coeliac patients. Moreover, when the Asp57-negative DQ beta chain is encoded from only one of the two DQB1 genes the DQA1*0501 allele is always present.     

套式聚合酶链式反应（Nested PCR）检测肺炎衣原体

肺炎衣原体（Ｃｈｌａｍｙｄｉａ ｐｎｅｕｍｏｎｉａｅ，ＣＰｎ）可引起人类急性呼吸道炎症，临床表现以肺炎为主，已在世界上许多国家和地区引起流行，本文介绍通过套式ＰＣＲ检测ＣＰｎ，共检测１０９例呼吸道感染者之咽拭标本，检出１３例阳性（阳性率１２％），提示ＣＰｎ的呼吸道感染有我国并不少见。实验结果表明，套式ＰＣＲ用于ＣＰｍ检测方法的建立，在敏感、特异、简便、快速的特点，具有良好的应用前景。     

High prevalence of human papillomaviruses in fresh frozen breast cancer samples

While the etiology of breast cancer remains enigmatic, some recent reports have examined the role of human papillomavirus (HPV) in breast carcinogenesis. The purpose of this study was to determine the prevalence of HPV in breast cancer tissue using PCR analysis and sequencing. Fifty-four (54) fresh frozen breast cancers samples that were removed from a cohort of breast cancer patients were analyzed. Samples were tested for HPV using comprehensive PCR primers, and in situ hybridization was performed on paraffin embedded tissue sections. Findings were correlated with clinical and pathological characteristics. The HPV DNA prevalence in the breast cancer samples was 50% (27/54) with sequence analysis indicating all cases to be positive for HPV-18 type. While HPV patients were slightly younger, no correlation was noted for menopausal status or family history. HPV positive tumors were smaller with earlier T staging and demonstrated lesser nodal involvement compared to HPV negative cancers. In situ hybridization analyses proved negative. The high proportion of HPV positive breast cancers detected in this series using fresh frozen tissues cannot be dismissed, however the role of HPV in breast carcinogenesis remains unclear and may ultimately be ascertained by monitoring future breast cancer incidence amongst women vaccinated against high risk HPV types.