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1.
Reconstructed ancestral sequences improve pathogen identification using resequencing DNA microarrays
Berthet N Deletoile A Passet V Kennedy GC Manuguerra JC Cole ST Brisse S 《PloS one》2010,5(12):e15243
We describe the benefit of using reconstructed ancestral sequences (RAS) on resequencing microarrays for rapid pathogen identification, with Enterobacteriaceae rpoB sequences as a model. Our results demonstrate a sharp improvement of call rate and accuracy when using RASs as compared to extant sequences. This improvement was attributed to the lower sequence divergence of RASs, which also expanded the sequence space covered by the microarray. Extension of this novel microarray design strategy to viruses, antimicrobial resistance elements or toxins is straightforward. 相似文献
2.
Nicholas P. Ambulos Jr. Lisa M. Schumaker Trevor J. Mathias Ruth White Jennifer Troyer David Wells Kevin J. Cullen 《Journal of biomolecular techniques》2016,27(2):46-52
Available clinical human papilloma virus (HPV) diagnostics for head and neck cancer have limited sensitivity and/or fail to define the HPV genotype. Common HPV genotyping assays are costly and labor intensive. We sought to develop a next-generation sequencing (NGS)-based HPV genotyping assay that was sensitive enough to work on formalin-fixed paraffin-embedded (FFPE) samples. We developed an ion torrent NGS HPV genotyping assay using barcoded HPV PCR broad-spectrum general primers 5+/6+ (BSGP)5+/6+. To validate genotype specificity and use in archived clinical FFPE tumor samples, we compared NGS HPV genotyping at 2 sequencing centers with typing by Roche Linear Array assay in 42 oropharyngeal and cervical cancer specimens representing 10 HPV genotypes, as well as HPV-negative cases. To demonstrate the detection of a broad range of HPV genotypes, we genotyped a cohort of 266 cervical cancers. A comparison of NGS genotyping of FFPE cancer specimens with genotyping by Linear Array showed concordant results in 34/37 samples (92%) at sequencing site 1 and 39/42 samples (93%) at sequencing site 2. Concordance between sites was 92%. Designed for use with 10 ng genomic DNA, the assay detected HPV using as little as 1.25 ng FFPE-derived genomic DNA. In 266 cervical cancer specimens, the NGS assay identified 20 different HPV genotypes, including all 13 carcinogenic genotypes. This novel NGS assay provides a sensitive and specific high-throughput method to detect and genotype HPV in a range of clinical specimens derived from FFPE with low per-sample cost. 相似文献
3.
为了探讨PCR-测序法在宫颈脱落细胞样品中人乳头瘤病毒 (Human papillomavirus, HPV) 临床检测中的应用价值,采用HPV通用引物PGMY09/11针对HPV L1区基因序列进行PCR扩增,并通过DNA测序法对HPV进行基因分型。对于混合感染样品,利用HPV型别特异性引物PCR的方法进行基因分型。325例临床样品中,228例为HPV阳性,其中66例为混合感染。共发现27种不同的HPV型别,其中HPV 16比例最多,其次是HPV 58和52。高危型HPV检出率随病变程度加重显著性增加 相似文献
4.
Karla Lopes Mandu de Campos Ana Paula Machado Flávia Gatto de Almeida Camila Mareti Bonin Thiago Theodoro Martins Prata Larissa Zatorre Almeida Cacilda Tezelli Junqueira Padovani Alda Maria Teixeira Ferreira Carlos Eurico dos Santos Fernandes Inês Aparecida Tozetti 《Memórias do Instituto Oswaldo Cruz》2014,109(3):352-355
Women infected with human papillomavirus (HPV) are at a higher risk of developing
cervical lesions. In the current study, self and clinician-collected vaginal and
cervical samples from women were processed to detect HPV DNA using polymerase chain
reaction (PCR) with PGMY09/11 primers. HPV genotypes were determined using
type-specific PCR. HPV DNA detection showed good concordance between self and
clinician-collected samples (84.6%; kappa = 0.72). HPV infection was found in 30%
women and genotyping was more concordant among high-risk HPV (HR-HPV) than low-risk
HPV (HR-HPV). HPV16 was the most frequently detected among the HR-HPV types. LR-HPV
was detected at a higher frequency in self-collected; however, HR-HPV types were more
frequently identified in clinician-collected samples than in self-collected samples.
HPV infections of multiple types were detected in 20.5% of clinician-collected
samples and 15.5% of self-collected samples. In this study, we demonstrated that the
HPV DNA detection rate in self-collected samples has good agreement with that of
clinician-collected samples. Self-collected sampling, as a primary prevention
strategy in countries with few resources, could be effective for identifying cases of
HR-HPV, being more acceptable. The use of this method would enhance the coverage of
screening programs for cervical cancer. 相似文献
5.
The accurate genotyping of human papillomavirus (HPV) is clinically important because the oncogenic potential of HPV is dependent on specific genotypes. Here, we described the development of a bead-based multiplex HPV genotyping (MPG) method which is able to detect 20 types of HPV (15 high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 and 5 low-risk HPV types 6, 11, 40, 55, 70) and evaluated its accuracy with sequencing. A total of 890 clinical samples were studied. Among these samples, 484 were HPV positive and 406 were HPV negative by consensus primer (PGMY09/11) directed PCR. The genotyping of 484 HPV positive samples was carried out by the bead-based MPG method. The accuracy was 93.5% (95% CI, 91.0-96.0), 80.1% (95% CI, 72.3-87.9) for single and multiple infections, respectively, while a complete type mismatch was observed only in one sample. The MPG method indiscriminately detected dysplasia of several cytological grades including 71.8% (95% CI, 61.5-82.3) of ASCUS (atypical squamous cells of undetermined significance) and more specific for high grade lesions. For women with HSIL (high grade squamous intraepithelial lesion) and SCC diagnosis, 32 women showed a PPV (positive predictive value) of 77.3% (95% CI, 64.8-89.8). Among women >40 years of age, 22 women with histological cervical cancer lesions showed a PPV of 88% (95% CI, 75.3-100). Of the highest risk HPV types including HPV-16, 18 and 31 positive women of the same age groups, 34 women with histological cervical cancer lesions showed a PPV of 77.3% (95% CI, 65.0-89.6). Taken together, the bead-based MPG method could successfully detect high-grade lesions and high-risk HPV types with a high degree of accuracy in clinical samples. 相似文献
6.
J.‐H. Lee, N.‐W. Lee, S.‐W. Hong, Y.‐S. Nam, J.‐W. Choi and Y.‐S. Kim Establishment of an efficient multiplex real‐time PCR assay for human papillomavirus genotyping in cervical cytology specimens: comparison with hybrid capture II Objective: To establish an efficient multiplex real‐time PCR assay for 15 human papillomavirus (HPV) genotypes, we designed multiplexing parameters and compared our PCR system with the hybrid capture (HC) II test using cervical cytology samples. Methods: For preventing cross‐reactive amplifications, variable HPV genes (E1, E2, E6, E7 and L1) were targeted. The melting temperatures of all primers and probes, and the size of the PCR product were optimized for the multiplex PCR. Our PCR system was compared with the HC II assays in the detection and genotyping of HPV infection using 173 cytology smears. Discordant cases between the two assays were verified by direct HPV DNA sequencing. Results: Of 173 women, 93 (53.8%) were HPV‐positive by the HC II assay and/or the multiplex real‐time PCR assay. The HPV genotypes were determined in 92 (98.9%) of 93 cases by the multiplex real‐time PCR and/or DNA sequencing. The agreement rate between multiplex PCR and HC II methods was 91.9% (kappa = 0.84). Although the sample size of this study needs to be increased to have epidemiological significance, multiple infections and HPV 16 were the predominant type. HPV 58, 52 and 18 accounted for 25% of HPV infections. HPV 52, 58 and 31 constituted 30% of CIN 2/3. Conclusion: The multiplex real‐time PCR system shows a good and reliable clinical performance. This in house PCR assay is fast and cost‐effective for HPV genotyping and the detection of HPV co‐infection in the post‐HPV vaccination era. 相似文献
7.
BackgroundThe objective of this work was to provide an update information on HPV age/genotype distribution by retrospectively analyzing a cohort of women living in the metropolitan area of Naples.MethodsFrom January 2011 to December 2017, cervical scrape specimens from 1265 women, with abnormal cytological indication, were tested for HPV DNA. The presence and the viral genotypes were assessed by the Linear Array HPV genotyping test for the detection of 37 anogenital HPV-DNA genotypes.ResultsThe overall prevalence of HPV infections was of 44.5% (95% CI 41.77–47.24). Among HR-HPV types, HPV-16 was the most common identified genotype, followed by HPV-31, -66, -59 and -51. As concern LR-HPV, HPV-53 resulted the most prevalent. Stratifying the study population by age, the total HPV infections showed a peak in younger women aged <23 years (58.5%), with a significative decrease by age (23–29 years, 54%; ≥ 30 years, 38.2%) (p < 0.001).ConclusionWe provided an HPV epidemiological analysis, highlighting the need to implement vaccination programmes and preventative screening strategies. 相似文献
8.
Oligonucleotide Microarray with RD-PCR Labeling Technique for Detection and Typing of Human Papillomavirus 总被引:4,自引:0,他引:4
Currently, screening for high-risk human papillomavirus (HPV) infection remains an important health concern throughout the
world, because of the close association between certain types of HPV and cervical cancer. In this study, we explore the possibility
of using ∼70mer oligonucleotide microarray for detection and genotyping of HPV. The ∼70mer type-specific oligonucleotide probes
of four different types HPV were designed by using biological software Arraydesigner 2.0, which analyzed the whole genome
sequences of HPV and selected optimal probes. These probes were synthesized and printed onto the surface of glass slides in
order to prepare a low-density microarray. HPV samples were labeled with fluorescence dyes Cy3 using a method of restriction
display polymerase chain reaction (RD-PCR). HPV plasmid DNA was restricted with Sau3A I to produce multiple fragments that
were ligated to adaptors subsequently and used as PCR template. PCR labeling was performed with the fluorescently labeled
universal primer (Cy3-UP) whose sequence is designed according to the adaptor of the RD-PCR approaches. The labeled samples
were hybridized with the oligonucleotide microarray. The scanning results showed that HPV DNA hybridized specifically with
multiple spots correspondingly to show positive signals, whereas no signals were detected of all the negative and blank controls.
These results demonstrated that ∼70mer oligonucleotide microarray can be applied to HPV detection and genotyping. The application
of RD-PCR in the sample labeling can increase significantly the sensitivity of the assay and will be especially useful for
the discriminate diagnosis of multiple pathogens. 相似文献
9.
Ritari J Hultman J Fingerroos R Tarkkanen J Pullat J Paulin L Kivi N Auvinen P Auvinen E 《PloS one》2012,7(3):e34211
Sensitive and specific detection of human papillomaviruses (HPV) in cervical samples is a useful tool for the early diagnosis of epithelial neoplasia and anogenital lesions. Recent studies support the feasibility of HPV DNA testing instead of cytology (Pap smear) as a primary test in population screening for cervical cancer. This is likely to be an option in the near future in many countries, and it would increase the efficiency of screening for cervical abnormalities. We present here a microarray test for the detection and typing of 15 most important high-risk HPV types and two low risk types. The method is based on type specific multiplex PCR amplification of the L1 viral genomic region followed by ligation detection reaction where two specific ssDNA probes, one containing a fluorescent label and the other a flanking ZipCode sequence, are joined by enzymatic ligation in the presence of the correct HPV PCR product. Human beta-globin is amplified in the same reaction to control for sample quality and adequacy. The genotyping capacity of our approach was evaluated against Linear Array test using cervical samples collected in transport medium. Altogether 14 out of 15 valid samples (93%) gave concordant results between our test and Linear Array. One sample was HPV56 positive in our test and high-risk positive in Hybrid Capture 2 but remained negative in Linear Array. The preliminary results suggest that our test has accurate multiple HPV genotyping capability with the additional advantages of generic detection format, and potential for high-throughput screening. 相似文献
10.
Cervical cancer is a leading cause of cancer-related deaths among women in India.Human papillomavirus (HPV) infection is the causative agent of cervical cancer; and infection with the high-risk genotypes, predominantly HPV16 and 18,is the biggest risk factor.Vaccines targeting HPV16 and 18 have been found to confer protection in large- scale clinical trials.HPV genotyping has traditionally been carried out to screen the population "at risk" using indirect methods based on polymerase chain reaction (PCR) using consensus primers combined with various DNA hybridization techniques,and often followed by the sequencing of candidate products.Recently,a high-throughput and direct method based on DNA sequencing has been described for HPV genotyping using multiplex pyrosequencing. We present a pilot study on HPV genotyping of cervical cancer and non-malignant cervical samples using multiplex pyrosequencing.Using genomic DNA from cell lines,cervical biopsies,surgical tissues or formalin-fixed,paraffin- embedded tissue samples,we could successfully resolve 6 different HPV types out of the 7 tested,with their prevalence found to be in agreement with earlier reports. We also resolved coinfections with two different HPV types in several samples. An HPV16 genotype with a specific and recurrent sequence variation was observed in 8 cancer samples and one non-malignant sample. We find this technique eminently suited for high-throughput applications,which can be easily extended to large sample cohorts to determine a robust benchmark for HPV genotypes prevalent in India. 相似文献
11.
E. Beerens L. Van Renterghem M. Praet Y. Sturtewagen S. Weyers M. Temmerman H. Depypere P. Claeys C. A. Cuvelier 《Cytopathology》2005,16(4):199-205
OBJECTIVES: In this study, we focus on the prevalence and occurrence of different anogenital human papillomavirus (HPV) genotypes in a first abnormal cervical screening test, and correlate HPV genotyping with the cytological diagnosis on thin-layer liquid-based preparations in routine gynaecological screening. METHODS: Out of 780 abnormal smears, 513 tested positive for HPV. All 25 different HPV types were identified by Line Probe Assay. RESULTS: The prevalence of high-risk HPV types increased from 72% in atypical squamous cell of undetermined significance to 94.5% in high-grade intra-epithelial lesion (HSIL). Co-infection with multiple HPV types was predominantly found in HSIL (35.8%). In the HSIL group the most common HPV types were 16, 52, 51 and 31; type 18 was rarely present. CONCLUSION: The role of types 31, 51 and 52 should be considered in future studies on vaccine development. 相似文献
12.
Campbell CD Sampas N Tsalenko A Sudmant PH Kidd JM Malig M Vu TH Vives L Tsang P Bruhn L Eichler EE 《American journal of human genetics》2011,(3):1102-332
Copy-number variants (CNVs) can reach appreciable frequencies in the human population, and recent discoveries have shown that several of these copy-number polymorphisms (CNPs) are associated with human diseases, including lupus, psoriasis, Crohn disease, and obesity. Despite new advances, significant biases remain in terms of CNP discovery and genotyping. We developed a method based on single-channel intensity data and benchmarked against copy numbers determined from sequencing read depth to successfully obtain CNP genotypes for 1495 CNPs from 487 human DNA samples of diverse ethnic backgrounds. This microarray contained CNPs in segmental duplication-rich regions and insertions of sequences not represented in the reference genome assembly or on standard SNP microarray platforms. We observe that CNPs in segmental duplications are more likely to be population differentiated than CNPs in unique regions (p = 0.015) and that biallelic CNPs show greater stratification when compared to frequency-matched SNPs (p = 0.0026). Although biallelic CNPs show a strong correlation of copy number with flanking SNP genotypes, the majority of multicopy CNPs do not (40% with r > 0.8). We selected a subset of CNPs for further characterization in 1876 additional samples from 62 populations; this revealed striking population-differentiated structural variants in genes of clinical significance such as OCLN, a tight junction protein involved in hepatitis C viral entry. Our microarray design allows these variants to be rapidly tested for disease association and our results suggest that CNPs (especially those that cannot be imputed from SNP genotypes) might have contributed disproportionately to human diversity and selection. 相似文献
13.
Background
Genotyping of human papillomarvirus (HPV) is crucial for patient management in a clinical setting. This study accesses the combined use of broad-range real-time PCR and high-resolution melting (HRM) analysis for rapid identification of HPV genotypes.Methods
Genomic DNA sequences of 8 high-risk genotypes (HPV16/18/39/45/52/56/58/68) were subject to bioinformatic analysis to select for appropriate PCR amplicon. Asymmetric broad-range real-time PCR in the presence of HRM dye and two unlabeled probes specific to HPV16 and 18 was employed to generate HRM molecular signatures for HPV genotyping. The method was validated via assessment of 119 clinical HPV isolates.Results
A DNA fragment within the L1 region was selected as the PCR amplicon ranging from 215–221 bp for different HPV genotypes. Each genotype displayed a distinct HRM molecular signature with minimal inter-assay variability. According to the HRM molecular signatures, HPV genotypes can be determined with one PCR within 3 h from the time of viral DNA isolation. In the validation assay, a 91% accuracy rate was achieved when the genotypes were in the database. Concomitantly, the HRM molecular signatures for additional 6 low-risk genotypes were established.Conclusions
This assay provides a novel approach for HPV genotyping in a rapid and cost-effective manner. 相似文献14.
Sun-Young Jun Eun Su Park Jiyoung Kim Jun Kang Jae Jun Lee Yoonjin Bae Sang-Il Kim Lee-So Maeng 《PloS one》2015,10(10)
Detecting high-risk (HR) HPV is important for clinical management of women with persistent HPV-positive and Pap-negative results. The Cobas 4800 HPV test is the first FDA-approved HPV DNA test that can be used alone as a first-line screening tool. The HPV 9G DNA chip test is a PCR-based DNA microarray assay. We evaluated the patients of consecutive HPV-positivity on HPV 9G DNA chip test without cytologic abnormalities. We then compared the performances of HPV 9G DNA chip and the Cobas 4800 HPV tests for detecting HR HPV with each other and confirmed HPV genotyping using direct sequencing. All 214 liquid-based cytology specimens were collected from 100 women with consecutive HPV-positive and Pap-negative results on the HPV 9G DNA chip test between May 2012 and Dec 2013, but only 180 specimens were available for comparing HPV test results. The HPV 9G DNA chip and the Cobas 4800 HPV tests agreed with each other in 81.7% of the samples, and the concordance rate was greater than 97.2% for detecting HPV-16 or -18. For HR genotypes other than HPV types 16 and 18, the two tests agreed for 81.1% of the samples. The sensitivity of both assays for detecting HR HPV was 100%, regardless of HR genotypes. The HPV 9G DNA chip test may be as effective as the Cobas 4800 HPV test in detecting HR HPV, and has a similar ability to identify HPV-16 and -18. 相似文献
15.
Objective: To ascertain the usefulness of the Roche Linear Array human papillomavirus (HPV) genotyping assay for assessing HPV genotypes in liquid-based cytology (LBC) samples and to evaluate this methodology within a cytopathology laboratory. These tests are of importance as persistent infection with high-risk HPV genotypes is considered a causal factor in the development of cervical cancer.
Methods: A total of 175 cervical LBC samples were tested using the Roche Linear Array HPV genotyping test. The suitability of the assay use in routine cytopathology laboratory was considered. HPV genotypes were matched to the cervical cytology results, which included negative, borderline nuclear abnormalities, mild, moderate and severe dyskaryosis.
Results: The assay could be applied to screening samples with the combined result available at the reporting stage. There were no test failures. All samples used after cytological analysis had sufficient DNA for testing. The results were reproducible and easily read and there was concordance of results between biomedical scientists. The results of the assay showed co-infection with multiple HPV genotypes was common in both high-grade and low-grade cytology samples. The percentage of HPV+ samples in the normal cytology samples (although in this grouping the number of samples was low) was 37%. In the cytology samples reported as severe dyskaryosis the HPV genotypes most commonly found were HPV16 and HPV51.
Conclusion: The assay was able to detect multiple HPV infection with a wide range of genotypes in LBC samples sent for routine cytological analysis. It would be suitable for use in a cytopathology laboratory. The results of the assay show that the genotype profile has some variation from other geographical regions, and more work is needed to determine population prevalence, to ascertain the impact of the HPV vaccine, to evaluate test for cure and HPV triage management. 相似文献
Methods: A total of 175 cervical LBC samples were tested using the Roche Linear Array HPV genotyping test. The suitability of the assay use in routine cytopathology laboratory was considered. HPV genotypes were matched to the cervical cytology results, which included negative, borderline nuclear abnormalities, mild, moderate and severe dyskaryosis.
Results: The assay could be applied to screening samples with the combined result available at the reporting stage. There were no test failures. All samples used after cytological analysis had sufficient DNA for testing. The results were reproducible and easily read and there was concordance of results between biomedical scientists. The results of the assay showed co-infection with multiple HPV genotypes was common in both high-grade and low-grade cytology samples. The percentage of HPV+ samples in the normal cytology samples (although in this grouping the number of samples was low) was 37%. In the cytology samples reported as severe dyskaryosis the HPV genotypes most commonly found were HPV16 and HPV51.
Conclusion: The assay was able to detect multiple HPV infection with a wide range of genotypes in LBC samples sent for routine cytological analysis. It would be suitable for use in a cytopathology laboratory. The results of the assay show that the genotype profile has some variation from other geographical regions, and more work is needed to determine population prevalence, to ascertain the impact of the HPV vaccine, to evaluate test for cure and HPV triage management. 相似文献
16.
Yi‐Chen Yang Der‐Yuan Wang Hwei‐Fang Cheng Eric Y. Chuang Mong‐Hsun Tsai 《Microbial biotechnology》2015,8(1):93-102
The genotyping of the hepatitis C virus (HCV) plays an important role in the treatment of HCV because genotype determination has recently been incorporated into the treatment guidelines for HCV infections. Most current genotyping methods are unable to detect mixed genotypes from two or more HCV infections. We therefore developed a multiplex genotyping assay to determine HCV genotypes using a bead array. Synthetic plasmids, genotype panels and standards were used to verify the target‐specific primer (TSP) design in the assay, and the results indicated that discrimination efforts using 10 TSPs in a single reaction were extremely successful. Thirty‐five specimens were then tested to evaluate the assay performance, and the results were highly consistent with those of direct sequencing, supporting the reliability of the assay. Moreover, the results from samples with mixed HCV genotypes revealed that the method is capable of detecting two different genotypes within a sample. Furthermore, the specificity evaluation results suggested that the assay could correctly identify HCV in HCV/human immunodeficiency virus (HIV) co‐infected patients. This genotyping platform enables the simultaneous detection and identification of more than one genotype in a same sample and is able to test 96 samples simultaneously. It could therefore provide a rapid, efficient and reliable method of determining HCV genotypes in the future. 相似文献
17.
Anogenital warts contain several distinct species of human papillomavirus. 总被引:10,自引:2,他引:8
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Anogenital warts from 26 patients were examined for the presence of human papillomavirus (HPV). Although no whole, intact virus could be identified, varying amounts of nonintegrated HPV DNA were detected in 18 tissue specimens (70%) by employing both an agarose gel-ethidium bromide staining method and the Southern blot hybridization procedure. When hybridization analysis was performed under stringent conditions, six anogenital warts were observed to contain HPV genomic sequences related to either of the cutaneous viruses HPV type 1 (HPV-1) or HPV-2. In 12 tissue samples lacking sequence homology to either HPV-1 or HPV-2 under stringent conditions, HPV-related sequences were detected when the hybridization was performed under less stringent conditions, indicating that an HPV distinct from both HPV-1 and HPV-2 is also associated with these lesions. This anogenital HPV also appeared to be distinct from the other characterized types of HPV. These data indicate that at least three HPVs are associated with anogenital wart disease. 相似文献
18.
Lixia Fu Chengcheng Cai Yinan Cui Jian Wu Jianli Liang Feng Cheng Xiaowu Wang 《Molecular breeding : new strategies in plant improvement》2016,36(4):48
Whole-genome resequencing (WGR) is a high-throughput way to determine genomic variations in breeding-related research. Accuracy and sensitivity are two of the most important issues in variation calling of WGR, especially for samples with low-depth resequencing data, which are used to reduce cost and save time in studies as survey of core germplasms from natural populations or genome-based breeding selection in segregation populations. An approach called pooled mapping was developed to call variations from low-depth resequencing data of natural or segregation populations. It is highly accurate and sensitive. First, pooled mapping creates a library of confident polymorphic loci in genomes of the population; then, the genotypes are called out at these confident loci for each sample in an efficient manner. The reliability of this pooled mapping method was confirmed using simulated datasets, real resequencing data and experimental genotyping. With onefold simulated resequencing data, results showed that pooled mapping identified SNPs in high accuracy (99.59 %) and sensitivity (93 %), compared to the commonly used method (accuracy: 29 %; sensitivity: 56 %). For the real low-depth resequencing data (≈0.8×) of 281 B. oleracea accessions, four loci corresponding to 1063 sites were selected for KASP genotyping to confirm the performance of pooled mapping. We found for all the 875 homozygous sites analyzed, pooled mapping achieved accuracy as 98.24 % and a sensitivity as 90.97 %. In conclusion, pooled mapping is an efficient means of determining reliable genomic variations with limited resequencing data for population samples. It will be a valuable tool in population genomic analysis and genome-based breeding research. 相似文献
19.
A highly specific microarray method for point mutation detection 总被引:1,自引:0,他引:1
Baaj Y Magdelaine C Ubertelli V Valat C Talini L Soussaline F Khomyakova E Funalot B Vallat JM Sturtz FG 《BioTechniques》2008,44(1):119-126
Improvements of microarray techniques for genotyping purposes have focused on increasing the reliability of this method. Here we report the development of a genotyping method where a microarray was spotted with stemloop probes, especially designed to optimize the hybridization specificity of complementary DNA sequences. This accurate method was used to screen for four common disease-causing mutations involved in a neurological disorder called Charcot-Marie-Tooth disease (CMT). Healthy individuals' and patients' DNA were amplified and labeled by PCR and hybridized on microarray. The spot signal intensities were 81 to 408 times greater for perfect compared with mismatched target sequences, differing by only one nucleotide (discrimination ratio) for healthy individual "homozygous" DNA. On the other hand, "heterozygous" mutant DNA samples gave rise to signal intensity ratios close to 1, as expected. The genotypes obtained by this method were perfectly consistent with those determined by direct PCR sequencing. Cross-hybridization rates were very low, resulting in further multiplexing improvements. In this study, we also demonstrated the feasibility of real-time hybridization detection of labeled synthetic oligonucleotides with concentrations as low as 2.5 nM. 相似文献
20.
目的 了解深圳地区妇女人群子宫颈感染HPV基因亚型流行特征。方法应用PCR结合反向寡核苷酸探针斑点杂交技术对1455例HPV阳性感染者进行基因亚型检测。结果1455例阳性感染者,单一型感染1277例,占87.8%,混合型感染178例,占12.2%;前5位胛y高危基因亚型分别是HPV16、52、58、18、33;在2006~2008年与2009~2010年两个时间段之间,HPV16、33和52基因亚型构成比明显不同,差异均具有统计学意义。结论子宫颈感染HPV以单一型为主,HPV16、52、58、18、33是感染的主要高危基因亚型;HPV高危基因亚型构成比在不同阶段存在差异。 相似文献