Consensus Primers Research Articles

Minimal Residual Disease in Multiple Myeloma

Minimal Residual Disease in Multiple Myeloma

Shewanella haliotisAssociated with Severe Soft Tissue Infection, Thailand, 2012

<i>Shewanella haliotis</i>Associated with Severe Soft Tissue Infection, Thailand, 2012

Comparison of two PCR strategies for the detection of bovine papillomavirus

Bovine papillomavirus (BPV) is a diverse group of double-stranded DNA oncogenic viruses, which have been detected in epithelial lesions and body fluids. Most studies of BPV infection rely on a single method for DNA detection; however the use of any single method or technique may underestimate the true prevalence of this virus. The purpose of this study was to compare two PCR strategies for the detection of BPV in skin lesions and fluids: these involve the use of BPV type-specific and consensus primers. Seventy-two cutaneous lesions, 57 blood samples and 59 semen samples were collected. PCR was used with the FAP consensus primers and BPV type-specific primers (for BPVs 2, 3, 4, 5, 8, 9 and 10), along with sequencing assays, to detect the BPV types. Phylogenetic analysis was carried out by means of the maximum likelihood method. It was found that both FAP and BPV type-specific primer sets could amplify BPV types of DNA in skin lesions, blood and semen samples. However, the BPV type-specific primers were more sensitive than the consensus primers and were able to detect co-infection of BPV in the samples. The consensus primers amplified five BPV types and were more suitable for detecting new putative BPV types. Thus, account should be taken of both PCR primer systems to identify co-infection, the presence of novel viruses, and avoid false-negative results.

미토콘드리아 DNA 염기서열 변이를 이용한 인삼 종 판별 연구

This study describes the identification of Panax species using mitochondrial consensus primers. Initially, a of thirty primers were tested in ten Korean ginseng cultivars and two foreign Panax species, P. quinquefolius and P. notogin-seng. In the polymerase chain reaction (PCR) amplification results, three primers (coxl, nadl/2-3 and nad2/l-2) generated co-dominant polymorphic handing patterns discriminating Korean ginseng cultivars from P. quinquefolius and P. notogin-seng. However these primers could not generated polymorphisms among the Korean ginseng cultivars, and simply represented species-specific polymorphisms for P. quinquefolius and P. notoginseng. Primers PQ9I and PN418 were designed from the consensus sequence of nad1/2-3 region. Two banding patterns (A or B) were detected in PQ9I. Korean ginseng and P. notoginseng shared the same banding pattern (A type) and P. quinquefolius was identified another banding (B type). In the case of PI

Human papillomavirus was not detected by PCR using multiple consensus primer sets in esophageal adenocarcinomas in Chinese patients

The role of human papillomavirus (HPV) infection in the development of esophageal squamous cell carcinoma is well established; however, there are few reports on the role of HPV in esophageal adenocarcinoma. To evaluate the putative role of HPV infection in esophageal adenocarcinoma, 57 formalin-fixed, paraffin-embedded esophageal adenocarcinoma specimens were collected from four hospitals in Shanghai and Anyang, China, between 1999 and 2008. HPV DNA was analyzed using PCR with multiple sets of consensus primers for HPV, GP5+/6+, CPI/CPIIG, SPF10, pU-1M/pU2R, and pU31B/pU2R. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the internal control, was amplified successfully in all 57 specimens. However, HPV amplification was not detected in any specimens with any of the consensus primer sets used. The present study indicates that HPV infection is not likely to be a major factor in the etiology of esophageal adenocarcinoma in the Chinese population.

Phylogenetic analysis of partial L1 gene of 10 human papillomavirus types isolated most commonly from women with normal and abnormal cervical cytology in Kuwait

This study was undertaken to evaluate the presence of human papillomavirus (HPV) variants in cervical samples. L1 genetic variable region was studied in 10 HPV types: HPV 11, 16, 18, 33, 53, 54, 56, 61, 66 and 81. A total of 116 isolates were examined, including 47 HPVs isolated from women with normal cytology and 69 with abnormal cytology of different grades. HPV sequences were detected using MY09/MY11 consensus primers. Fifty silent and 65 missense mutations were detected. Two missense mutations were detected in HPV18, 3 in HPV56 and 17 in HPV61. The number of missense mutations per isolate ranged from 1 to 3, except in HPV54 and HPV61, where 7 and 11 missense mutations were found, respectively. Most of the isolates (52.3%) with missense mutations were isolated from women with abnormal cervical samples. Low-grade squamous intraepithelial lesion cytology diagnosis dominated all cervical abnormalities. This study is the first on the identification of molecular variants in the Middle East and suggests the circulation of new HPV subtypes and variants in Kuwait, which needs to be confirmed by further analysis of the complete HPV genome.

Mitochondrial DNA Copy Number and Risk of Oral Cancer: A Report from Northeast India

BackgroundOral squamous cell carcinoma (OSCC) is the sixth most common cancer globally. Tobacco consumption and HPV infection, both are the major risk factor for the development of oral cancer and causes mitochondrial dysfunction. Genetic polymorphisms in xenobiotic-metabolizing enzymes modify the effect of environmental exposures, thereby playing a significant role in gene–environment interactions and hence contributing to the individual susceptibility to cancer. Here, we have investigated the association of tobacco - betel quid chewing, HPV infection, GSTM1-GSTT1 null genotypes, and tumour stages with mitochondrial DNA (mtDNA) content variation in oral cancer patients.Methodology/Principal FindingsThe study comprised of 124 cases of OSCC and 140 control subjects to PCR based detection was done for high-risk HPV using a consensus primer and multiplex PCR was done for detection of GSTM1-GSTT1 polymorphism. A comparative ΔCt method was used for determination of mtDNA content. The risk of OSCC increased with the ceased mtDNA copy number (Ptrend = 0.003). The association between mtDNA copy number and OSCC risk was evident among tobacco – betel quid chewers rather than tobacco – betel quid non chewers; the interaction between mtDNA copy number and tobacco – betel quid was significant (P = 0.0005). Significant difference was observed between GSTM1 - GSTT1 null genotypes (P = 0.04, P = 0.001 respectively) and HPV infection (P<0.001) with mtDNA content variation in cases and controls. Positive correlation was found with decrease in mtDNA content with the increase in tumour stages (P<0.001). We are reporting for the first time the association of HPV infection and GSTM1-GSTT1 null genotypes with mtDNA content in OSCC.ConclusionOur results indicate that the mtDNA content in tumour tissues changes with tumour stage and tobacco-betel quid chewing habits while low levels of mtDNA content suggests invasive thereby serving as a biomarker in detection of OSCC.

Minimal residual disease quantification using consensus primers and high-throughput IGH sequencing predicts post-transplant relapse in chronic lymphocytic leukemia

Quantification of minimal residual disease (MRD) following allogeneic hematopoietic cell transplantation (allo-HCT) predicts post-transplant relapse in patients with chronic lymphocytic leukemia (CLL). We utilized an MRD-quantification method that amplifies immunoglobulin heavy chain (IGH) loci using consensus V and J segment primers followed by high-throughput sequencing (HTS), enabling quantification with a detection limit of one CLL cell per million mononuclear cells. Using this IGH–HTS approach, we analyzed MRD patterns in over 400 samples from 40 CLL patients who underwent reduced-intensity allo-HCT. Nine patients relapsed within 12 months post-HCT. Of the 31 patients in remission at 12 months post-HCT, disease-free survival was 86% in patients with MRD <10−4 and 20% in those with MRD ⩾10−4 (relapse hazard ratio (HR) 9.0; 95% confidence interval (CI) 2.5–32; P<0.0001), with median follow-up of 36 months. Additionally, MRD predicted relapse at other time points, including 9, 18 and 24 months post-HCT. MRD doubling time <12 months with disease burden ⩾10−5 was associated with relapse within 12 months of MRD assessment in 50% of patients, and within 24 months in 90% of patients. This IGH–HTS method may facilitate routine MRD quantification in clinical trials.

Isolation and characterization of multiple F-box genes linked to the S 9 - and S 10 -RNase in apple (Malus × domestica Borkh.)

Using 11 consensus primer pairs designed from S-linked F-box genes of apple and Japanese pear, 10 new F-box genes (MdFBX21 to 30) were isolated from the apple cultivar 'Spartan' (S(9)S(10)). MdFBX21 to 23 and MdFBX24 to 30 were completely linked to the S(9) -RNase and S(10-)RNase, respectively, and showed pollen-specific expression and S-haplotype-specific polymorphisms. Therefore, these 10 F-box genes are good candidates for the pollen determinant of self-incompatibility in apple. Phylogenetic analysis and comparison of deduced amino acid sequences of MdFBX21 to 30 with those of 25 S-linked F-box genes previously isolated from apple showed that a deduced amino acid identity of greater than 88.0 % can be used as the tentative criterion to classify F-box genes into one type. Using this criterion, 31 of 35 F-box genes of apple were classified into 11 types (SFBB1-11). All types included F-box genes derived from S(3-) and S(9-)haplotypes, and seven types included F-box genes derived from S(3-), S(9-), and S(10-)haplotypes. Moreover, comparison of nucleotide sequences of S-RNases and multiple F-box genes among S(3-), S(9-), and S(10-)haplotypes suggested that F-box genes within each type showed high nucleotide identity regardless of the identity of the S-RNase. The large number of F-box genes as candidates for the pollen determinant and the high degree of conservation within each type are consistent with the collaborative non-self-recognition model reported for Petunia. These findings support that the collaborative non-self-recognition system also exists in apple.

Association between age and high‐risk human papilloma virus in Mexican oral cancer patients

Studies reporting low prevalence of HPV in OSCC with declining age at presentation are increasing. The aim of this study was to determine the prevalence of HPV in a group of OSCC cases and controls in a Mexican population. The matched case-control study included 80 OSCC cases and 320 controls. HPV/DNA presence was evaluated through PCR amplification using three sets of consensus primers for the L1 gene. A conditional logistic regression analysis was carried out for the matched OSCC cases and controls. Interactions between risk factors and OCSS were tested in the construction process of the models. HPV prevalence was 5% in OSCC cases and 2.5% in controls. HPV-detected types were 16, 18 and 56. According to conditional logistics regression model, an association was detected between HR-HPV and OSCC. All HR-HPV-positive OSCC cases corresponded to young patients (<45years), non-smokers and non-alcohol drinkers. The HR-HPV can be a contributing factor to oral carcinogenesis, especially in younger individuals without known risk factors such as tobacco and alcohol.

Papillomaviral DNA sequences are not amplifiable from canine subungual squamous cell carcinomas

AIM: To determine if papillomaviral DNA is more frequently present within canine subungual squamous cell carcinomas (SCCs) than in non-SCC digit lesions. METHODS: Total DNA was extracted from 23 canine subungual SCCs and 23 non-SCC digit lesions. The presence of amplifiable DNA within each sample was confirmed by amplifying a section of the glyceraldehyde-3-phosphate dehydrogenase (GADPH) gene. Two different consensus PCR primer sets were used to amplify papillomaviral DNA from the samples. RESULTS: The consensus primers only amplified papillomaviral DNA from the positive control samples. None of the 46 canine digit samples contained DNA that was amplifiable by the consensus PCR primers. CONCLUSION: Papillomaviruses are unlikely to be a significant cause of canine subungual SCCs. CLINICAL RELEVANCE: While circumstantial evidence suggests that canine subungual SCCs could develop due to papillomaviral infection, this study did not reveal any evidence to support papillomaviral aetiology of these neoplasms.

Elevated CD40 ligand silences α interferon production in an HIV-related immune reconstitution inflammatory syndrome

Elevated CD40 ligand silences α interferon production in an HIV-related immune reconstitution inflammatory syndrome

Cloning an S-RNase allele, including the longest intron, from cultivars of European pear (Pyrus communis L.)

SummaryMost cultivars of European pear (Pyrus communis L.) exhibit S-RNase-based self-incompatibility. Their S-genotypes have mainly been assigned by analysing PCR-amplified S-RNase alleles from the genomic DNA of each cultivar. In this study, we identified eight European pear cultivars that had only one S-RNase allele amplified by conventional genomic PCR using a consensus primer pair. Rapid amplification of cDNA ends (RACE) on stylar RNAs of the cultivars was used to obtain the full-length sequence of the putative S25-RNase. Long-PCR successfully amplified the S25-RNase allele, including a long 3,131 bp intron, the longest intron reported so far among S-RNase alleles in fruit tree species in the sub-tribe Pyrinae. Conventional PCR using a consensus primer and a primer designed from the intron sequence amplified a 385 bp fragment of the S25-RNase allele from the genomic DNA of all eight cultivars. An S-RNase-based, cleaved amplified polymorphic sequence (CAPS) marker system and S25-RNase allele-specific PCR were used to assign the eight pear cultivars to five genotypes. In addition, cDNAs of the S118- and S119-RNase alleles were re-cloned and sequenced to correct previously published sequences.

Molecular characterization of AfuFleA, an l-fucose-specific lectin from Aspergillus fumigatus

Aspergillus fumigatus causes life-threatening infections in immunocompromised patients. We have found that extracts of mycelial mats of A. fumigatus contain a potent hemagglutinin. To clarify the characteristics of this factor, the hemagglutinin was purified from late-stage cultures and characterized at the molecular level. The hemagglutinin is a 32-kilodalton protein that shows activity as an L-fucose lectin. The gene encoding this protein, AfufleA, was identified from a genomic DNA library utilizing consensus primers designed for amino acid sequences obtained from peptides following limited trypsin proteolysis. An open reading frame was found that consists of 942 nucleotides encoding 314 amino acids with a deduced molecular mass of 34,498 and contains all seven of trypsin-digested peptide sequences; four short introns, 49-63bp, were also identified. AfufleA shares homology with a fucose-specific lectin produced by the orange peel mushroom, Aleuria aurantia. The role of AfufleA fucose-specific lectin is not clear, but this lectin may enhance attachment of fungal spores to mammalian cell membranes and contribute to the pathogenicity of A. fumigatus.

A newly developed consensus polymerase chain reaction to detect Mycoplasma species using 16S ribosomal RNA gene

Mycoplasmas are highly fastidious bacteria, difficult to culture and slow growing. Infections with Mycoplasma species can cause a variety of problems in living organisms and in vitro cell cultures. In this study, we investigated the usefulness of a genus-specific consensus PCR analysis method to detect Mycoplasma species. The developed consensus primer pairs MycoF and MycoR were designed specifically to amplify the 16S ribosomal RNA gene (rRNA) of Mycoplasma species by the optimized PCR system. The developed consensus PCR system effectively amplified 215 bp of Mycoplasma genus-specific region of 16S rRNA. In conclusion, we recommend this consensus PCR for monitoring Mycoplasma species in animals, human and cell culture system.

High-Risk and Low-Risk Human Papillomavirus in Esophageal Squamous Cell Carcinoma at Mazandaran, Northern Iran

Cancers are the second most common cause of non-accidental deaths in Iran, following cardiovascular deaths. Mazandaran, near the Caspian Littoral at north of Iran have identified as a several-high incidence area for Esophageal Squamous Cell Carcinoma (ESCC) in the world. Several associated risk factors, such as dietary and cultural habits, infectious agents, nutritional deficiencies, too much use of tobacco and alcohol and infection to certain DNA tumor viruses (HPVs), including environmental and genetic factors are attributed to this disease. To explore this issue, we analyzed HPV DNA prevalence and HPV types together in relation to tumor sites a high-incidence population. Archived tissue blocks from 46, 69 and 62 upper, middle and lower third of esophagus, respectively from ESCC patients were evaluated for the presence of HPV DNA by PCR using the degenerate HPV L1 consensus primer pairs MY09/MY11. The positive specimens were evaluated by Real-time PCR to determine HPV genotypes. From the 49 HPV positive cases, of ESCC patients, 5 (23.1%), 11 (55%) and 9 (56.3%) of upper, middle and lower third of ESCC specimens, respectively were positive by at least one high and one low-risk HPV genotypes. In general, HPV45 and HPV11 were the most common high- risk and low-risk HPV genotypes in HPV L1 positive cases, respectively, followed by HPV6, HPV52 and HPV39. Therefore, the high prevalence of HPV DNA in different anatomical sites of ESCC patients from the Mazandaran region in North of Iran provides more evidence for a role of HPV in this cancer.

Cloning, Molecular Characterization, and mRNA Expression of the Thermostable Family 3 β-Glucosidase from the Rare Fungus Stachybotrys microspora

The filamentous fungus Stachybotrys microspora possess a rich β-glucosidase system composed of five β-glucosidases. Three of them were already purified to homogeneity and characterized. In order to isolate the β-glucosidase genes from S. microspora and study their regulation, a PCR strategy using consensus primers was used as a first step. This approach enabled the isolation of three different fragments of family 3 β-glucosidase gene. A representative genomic library was constructed and probed with one amplified fragment gene belonging to family 3 of β-glucosidase. After two rounds of hybridization, seven clones were obtained and the analysis of DNA plasmids leads to the isolation of one clone (CF3) with the largest insert of 7 kb. The regulatory region shows multiple TC-rich elements characteristic of constitutive promoter, explaining the expression of this gene under glucose condition, as shown by zymogram and RT-PCR analysis. The tertiary structure of the deduced amino acid sequence of Smbgl3 was predicted and has shown three conserved domains: an (α/β)8 triose phosphate isomerase (TIM) barrel, (α/β)5 sandwich, and fibronectin type III domain involved in protein thermostability. Zymogram analysis highlighted such thermostable character of this novel β-glucosidase.

Inverted papilloma of the ureter: study of a rare case with emphasis on clinicopathologic implications

A 62-year old male patient presented complaining of intermittent macroscopic hematuria. The ultrasonographic investigation revealed a hydronephrosis of remarkable degree with indiscreterenal parenchyma. The abdominal computed tomography scan identified a ureteral lesion with proximal dilatation, hydronephrosis and a functionless ipsilateral renal unit. The retrograde urography showed a 4-cm lesion with multiple filling defects and a smooth contour. The endoscopic examination showed an exophytic lesion, highly suspicious for malignancy. Urine cytology revealed atypia. Right nephroureterectomy was performed and the pathology revealed a ureteral inverted papilloma (UIP). Polymerase chain reaction examination for the presence of human papilloma virus, using GP5+/6+ consensus primers, was negative. The presence UIP should be considered in patients with urotheleal lesions in the ureter when the diagnostic workup for malignancy is inconclusive. The clinical course of the disease seems to be favorable.

Inverted papilloma of the ureter: study of a rare case with emphasis on clinicopathologic implications.

A 62-year old male patient presented complaining of intermittent macroscopic hematuria. The ultrasonographic investigation revealed a hydronephrosis of remarkable degree with indiscrete renal parenchyma. The abdominal computed tomography scan identified a ureteral lesion with proximal dilatation, hydronephrosis and a functionless ipsilateral renal unit. The retrograde urography showed a 4-cm lesion with multiple filling defects and a smooth contour. The endoscopic examination showed an exophytic lesion, highly suspicious for malignancy. Urine cytology revealed atypia. Right nephroureterectomy was performed and the pathology revealed a ureteral inverted papilloma (UIP). Polymerase chain reaction examination for the presence of human papilloma virus, using GP5+/6+ consensus primers, was negative. The presence UIP should be considered in patients with urotheleal lesions in the ureter when the diagnostic workup for malignancy is inconclusive. The clinical course of the disease seems to be favorable.

Salivary Mucoepidermoid Carcinoma: Demonstration of Transcriptionally Active Human Papillomavirus 16/18

Herein we test the following hypotheses: (1) High-risk Human Papillomavirus (HR-HPV) may be involved in the etiology of mucoepidermoid carcinoma (MEC), and (2) The detection rate of HR-HPV in MEC has been increasing over time. Ninety-eight archival MEC specimens from three institutions spanning three decades were studied for HPV16/18 E6/E7 transcripts. RNA was extracted from formalin-fixed paraffin embedded specimens and HPV16/18 E6/E7 expression assessed by nested reverse transcription polymerase chain reaction (RT-PCR). A subset of MEC were also studied for MECT1-MAML2 fusion transcripts by nested RT-PCR and amplicon sequencing. The HPV expression data was validated by immunofluorescence (IF) with monoclonal HPV16/18 E6 antibody, PCR with the GP5+/6+ consensus primers, and sequencing of RT-PCR amplicons. HPV genome was localized by in-situ hybridization with the Ventana Inform HPVIII Family 16 probe. P16(INK4a) overexpression and aberrant p53 expression were assessed by immunohistochemistry. HPV16 E6/E7 transcripts were demonstrated in (29/98) 30% of MEC by RT-PCR. HPV18 E6/E7 transcripts were demonstrated in 13/98 (13%) of MEC by RT-PCR. Seven of 98 tumors (7%) demonstrated both HPV16/18. No significant association was found between HPV status and gender, age, and tumor site. All 13 HPV18+ MEC were diagnosed between 2001 and 2010, whereas 45 MEC diagnosed from 1977 to 2000 were negative for HPV18 (p = 0.002). By contrast, there was no significant difference with respect to HPV16 detection and date of diagnosis. All MEC that were positive for E6 protein were also HPV16/18 positive by RT-PCR. Sequencing a subset of RT-PCR amplicons confirmed HPV type- and region-specific sequences. PCR using GP5+/6+ consensus primers demonstrated HPV status concordance in 9 of 10 cases. DNA degradation was present in the last case; the RT-PCR amplicons were sequenced from this case which confirmed the presence of HPV type- and region-specific sequences. Strong (+4/+4) and diffuse (>50%) nuclear and cytoplasmic p16 expression was seen in 64% of MEC in the glandular regions, and 18% of MEC in the solid, squamoid regions. No correlation was seen between p16 expression and HPV status. Twenty-nine MEC (22 HPV+ and 7 HPV-negative) were selected for further evaluation for p53 expression. Strong aberrant nuclear p53 expression was present in only 2/22 HPV + MEC (9%, both Grade 3); no HPV-negative MEC demonstrated aberrant p53 expression. MECT1-MAML2 fusion transcripts were demonstrated in 23/37 (62%) MEC. No significant association was found between the presence of the MECT1-MAML2 fusion transcripts and tumor grade, HPV status, gender, era of diagnosis (2000 and earlier vs. 2001-2010) or tumor site. We demonstrate for the first time that transcriptionally active HPV16/18 is common to MEC. These findings were validated by demonstrating concordant results by separate PCR with consensus primers, and/or confirming the presence of HPV type- and region-specific sequences in the RT-PCR amplicons. We also visualized E6 viral oncoprotein and HPV genome within tumor cells. HR-HPV is thus potentially implicated in the pathogenesis of MEC. The frequency of HPV18 detection is significantly increased in MEC diagnosed after 2001, whereas we found no differences in the HPV16 detection rates per era of diagnosis.