Generating DNA profile from low copy number DNA: Strategies and associated risks

Many shreds of evidence found on the crime scenes contain a trace amount of DNA which results in insignificant profiling results for subsequent comparison. This can nullify the potential evidence material and hamper investigation process. Over the years, different strategies have been employed by various DNA testing laboratories to create interpretable DNA profiles generated from low template of DNA. This review highlights different strategies used by forensic laboratories worldwide for creating complete DNA profiles from low copy number template for comparison purposes along with its associated risks for forensic purposes.

Characterization of clinical isolates of Mycobacterium tuberculosis from indigenous peoples of Colombia / Caracterización de aislamientos clínicos de Mycobacterium tuberculosis de pueblos indígenas de Colombia

ABSTRACT Introduction: Tuberculosis continues to be a public health priority. Indigenous peoples are vulnerable groups with cultural determinants that increase the risk of the disease. Objective: To determine molecular epidemiology and phenotypical features and of Mycobacterium tuberculosis isolates from indigenous people in Colombia during the period from 2009 to 2014. Materials and methods: We conducted an analytical observational study; we analyzed 234 isolates to determine their patterns of sensitivity to antituberculosis drugs and their molecular structures by spoligotyping. Results: The isolates came from 41 indigenous groups, predominantly the Wayúu (13.10%) and Emberá Chamí (11.35%). We found 102 spoligotypes distributed among seven genetic families (37.2% LAM, 15.8% Haarlem, 8.1% T, 3.4% U, 2.6% S, 2.1% X, and 0.9%, Beijing). The association analysis showed that the non-clustered isolates were related to prior treatment, relapse, orphan spoligotypes, and the Beijing family. The H family presented an association with the Arhuaco and Camëntŝá indigenous groups, the U family was associated with the Wounaan group, and the T family was associated with the Motilón Barí group. Conclusions: This is the first national study on M. tuberculosis characterization in indigenous groups. The study evidenced that diagnosis in indigenous people is late. We described 53% of orphan patterns that could be typical of the Colombian indigenous population. The high percentage of grouping by spoligotyping (62%) could indicate cases of active transmission, a situation that should be corroborated using a second genotyping marker. A new Beijing spoligotype (Beijing-like SIT 406) was identified in Colombia.

RESUMEN Introducción. La tuberculosis es prioridad de salud pública. Los pueblos indígenas son vulnerables debido a los factores culturales determinantes que aumentan el riesgo de tuberculosis. Objetivo. Determinar la epidemiologia molecular y las características fenotípicas de los aislamientos de Mycobacterium tuberculosis de pueblos indígenas de Colombia entre 2009 y 2014. Materiales y métodos. Se hizo un estudio observacional analítico; se analizaron 234 aislamientos para determinar la sensibilidad a los fármacos antituberculosos y la estructura molecular usando spoligotyping. La información epidemiológica se recolectó utilizando el formato único de vigilancia de micobacterias. Resultados. Los aislamientos provenían de 41 grupos indígenas, principalmente los wayúu (13,10 %) y emberá chamí (11,35 %). Se encontraron 102 genotipos distribuidos en siete familias genéticas (37,2 %, LAM; 15,8 %, Haarlem; 8,1 %, T; 3,4 %, U; 2,6 %, S; 2,1 %, X, y 0,9%, Beijing). El análisis de asociación mostró que los aislamientos no agrupados se asociaron con el tratamiento previo, las recaídas, los genotipos huérfanos y la familia Beijing. La familia H presentó una asociación con los grupos indígenas arhuaco y camëntŝá, la familia U se asoció con el grupo wounaan y la familia T con el grupo motilón barí. Conclusiones. Este es el primer estudio nacional de caracterización de M. tuberculosis en grupos indígenas. Se evidenció que el diagnóstico en indígenas es tardío, y que 53 % de los patrones huérfanos podrían ser típicos de la población indígena colombiana. El alto porcentaje de agrupamiento por spoligotyping (62%) podría indicar casos de transmisión activa, una situación que debe ser corroborada usando un segundo marcador de genotipificación. Se identificó un nuevo genotipo (Beijing-like SIT 406) en Colombia.

Global Quantitative Mapping of Enhancers in Rice by STARR-seq / 基因组蛋白质组与生物信息学报·英文版

Enhancers activate transcription in a distance-, orientation-, and position-independent manner, which makes them difficult to be identified. Self-transcribing active regulatory region sequencing (STARR-seq) measures the enhancer activity of millions of DNA fragments in parallel. Here we used STARR-seq to generate a quantitative global map of rice enhancers. Most enhancers were mapped within genes, especially at the 5' untranslated regions (5'UTR) and in coding sequences. Enhancers were also frequently mapped proximal to silent and lowly-expressed genes in transposable element (TE)-rich regions. Analysis of the epigenetic features of enhancers at their endogenous loci revealed that most enhancers do not co-localize with DNase I hypersensitive sites (DHSs) and lack the enhancer mark of histone modification H3K4me1. Clustering analysis of enhancers according to their epigenetic marks revealed that about 40% of identified enhancers carried one or more epigenetic marks. Repressive H3K27me3 was frequently enriched with positive marks, H3K4me3 and/or H3K27ac, which together label enhancers. Intergenic enhancers were also predicted based on the location of DHS regions relative to genes, which overlap poorly with STARR-seq enhancers. In summary, we quantitatively identified enhancers by functional analysis in the genome of rice, an important model plant. This work provides a valuable resource for further mechanistic studies in different biological contexts.

Evidence for regulated expression of Telomeric Repeat-containing RNAs (TERRA) in parasitic trypanosomatids

The Telomeric Repeat-containing RNAs (TERRA) participate in the homeostasis of telomeres in higher eukaryotes. Here, we investigated the expression of TERRA in Leishmania spp. and Trypanosoma brucei and found evidences for its expression as a specific RNA class. The trypanosomatid TERRA are heterogeneous in size and partially polyadenylated. The levels of TERRA transcripts appear to be modulated through the life cycle in both trypanosomatids investigated, suggesting that TERRA play a stage-specific role in the life cycle of these early-branching eukaryotes.

Study on Causes of Respiratory Disease to the Middle East Respiratory Syndrome-negative Subjects

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe cases of human respiratory disease. The current outbreak of infection with this virus in South Korea, which began on May 20, 2015, has infected 186 patients and caused 36 deaths within 2 months. In this study, to investigate the viral pathogen causing acute respiratory infections, multiplex/RT-PCR was performed on were obtained from nucleic acid of the Middle East Respiratory Syndrome-negative subjects. Viruses and atypical bacteria were detected in 39 of 337 (11.6%). Frequent viruses were human rhinovirus (n=11, 3.3%), human metapneumovirus (n=9, 2.7%), parainfluenza (n=9, 2.7%) and adenovirus (n=4, 1.2%). Mycoplasma pneumonia (M. pneumonia) was detected in 1.8 % (n=6). Out of 9 human metapneumovirus (hMPV) positive samples, 6 samples were successfully sequenced using F gene. And M. pneumoniae was sequencing of a repetitive region of the P1 gene. Phylogenetic analysis revealed that hMPV clustered into A2b lineage (n=4), B2 lineage (n=2) and M. pneumoniae clustered into two genotypes: Type 1 (n=4), Type 2a (n=2).

Study on Causes of Respiratory Disease to the Middle East Respiratory Syndrome-negative Subjects

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe cases of human respiratory disease. The current outbreak of infection with this virus in South Korea, which began on May 20, 2015, has infected 186 patients and caused 36 deaths within 2 months. In this study, to investigate the viral pathogen causing acute respiratory infections, multiplex/RT-PCR was performed on were obtained from nucleic acid of the Middle East Respiratory Syndrome-negative subjects. Viruses and atypical bacteria were detected in 39 of 337 (11.6%). Frequent viruses were human rhinovirus (n=11, 3.3%), human metapneumovirus (n=9, 2.7%), parainfluenza (n=9, 2.7%) and adenovirus (n=4, 1.2%). Mycoplasma pneumonia (M. pneumonia) was detected in 1.8 % (n=6). Out of 9 human metapneumovirus (hMPV) positive samples, 6 samples were successfully sequenced using F gene. And M. pneumoniae was sequencing of a repetitive region of the P1 gene. Phylogenetic analysis revealed that hMPV clustered into A2b lineage (n=4), B2 lineage (n=2) and M. pneumoniae clustered into two genotypes: Type 1 (n=4), Type 2a (n=2).

Genotyping of multidrug-resistant strains of Pseudomonas aeruginosa isolated from burn and wound infections by ERIC-PCR

ABSTRACT PURPOSE: To determine the genetic diversity of MDR P. aeruginosa strains isolated from burn and wound infections in Ahvaz, Iran, by ERIC-PCR. METHODS: From total 99 strains of P. aeruginosa defined as MDR by using drug susceptibility testing, 66 were subjected to ERIC-PCR analysis, comprises 53 strains isolated from burn infection, and 13 randomly selected strains from wound infection with higher resistance to combinations of more numbers of drugs. RESULTS: Eight clusters (I to VIII), and 50 single clones were generated for tested MDR isolates analyzed by ERIC-PCR. The high heterogeneity was observed among the isolates from burn infections including 16 isolates which were categorized in eight clusters and 37 single clones. The isolates in clusters II, III, VI, VIII showed 100% similarity. CONCLUSIONS: The high level of genotypic heterogeneity in P. aeruginosa strains demonstrated no genetic correlation between them. Extremely high drug resistance in isolates from burn, suggests that efficient control measures and proper antibiotic policy should be observed.

Prevalence of Trichomoniasis by PCR in Women Attending Health Screening in Korea

Trichomoniasis is the most common curable sexually-transmitted infection (STI) worldwide. There are few reports on the prevalence of Trichomonas vaginalis in Korea. The purpose of this study was to examine the prevalence of trichomoniasis by PCR in Guri city, Korea. All adult women who visited Hanyang University Guri Hospital for health screening within the National Health Care Service were invited to participate in the study, and 424 women were enrolled between March and June 2011. PCR was used to detect Trichomonas vaginalis using primers based on a repetitive sequence cloned from T. vaginalis (TV-E650). Fourteen women (3.3%) were found to have T. vaginalis. All were over 50, and they were significantly older on average than the 410 Trichomonas-negative women (mean ages 63.4 vs 55.3 years). It seems that T. vaginalis infection is not rare in women receiving health screening, especially among those over 50.

Molecular Strain Typing of Mycobacterium tuberculosis: a Review of Frequently Used Methods

Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, remains one of the most serious global health problems. Molecular typing of M. tuberculosis has been used for various epidemiologic purposes as well as for clinical management. Currently, many techniques are available to type M. tuberculosis. Choosing the most appropriate technique in accordance with the existing laboratory conditions and the specific features of the geographic region is important. Insertion sequence IS6110-based restriction fragment length polymorphism (RFLP) analysis is considered the gold standard for the molecular epidemiologic investigations of tuberculosis. However, other polymerase chain reaction-based methods such as spacer oligonucleotide typing (spoligotyping), which detects 43 spacer sequence-interspersing direct repeats (DRs) in the genomic DR region; mycobacterial interspersed repetitive units–variable number tandem repeats, (MIRU-VNTR), which determines the number and size of tandem repetitive DNA sequences; repetitive-sequence-based PCR (rep-PCR), which provides high-throughput genotypic fingerprinting of multiple Mycobacterium species; and the recently developed genome-based whole genome sequencing methods demonstrate similar discriminatory power and greater convenience. This review focuses on techniques frequently used for the molecular typing of M. tuberculosis and discusses their general aspects and applications.

Mutational and acquired carbapenem resistance mechanisms in multidrug resistant Pseudomonas aeruginosa clinical isolates from Recife, Brazil

An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem-resistant Pseudomonas aeruginosaisolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosaisolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXY-OprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed.

Resurgence of pertussis at the age of vaccination: clinical, epidemiological, and molecular aspects / Ressurgimento da coqueluche na era vacinal: aspectos clínicos, epidemiológicos e moleculares

OBJECTIVE: Report the incidence, epidemiology, clinical features, death, and vaccination status of patients with whooping cough and perform genotypic characterization of isolates of B. pertussis identified in the state of Paraná, during January 2007 to December 2013.METHODS: Cross-sectional study including 1,209 patients with pertussis. Data were obtained through the Notifiable Diseases Information System (Sistema de Informação de Agravos de Notificação - SINAN) and molecular epidemiology was performed by repetitive sequence-based polymerase chain reaction (rep-PCR; DiversiLab(r), bioMerieux, France).RESULTS: The incidence of pertussis in the state of Paraná increased sharply from 0.15-0.76 per 100,000 habitants between 2007-2010 to 1.7-4.28 per 100,000 between 2011-2013. Patients with less than 1 year of age were more stricken (67.5%). Fifty-nine children (5%) developed pertussis even after receiving three doses and two diphtheria-tetanus-pertussis (DTP) boosters vaccine. The most common complications were pneumonia (14.5%), otitis (0.9%), and encephalopathy (0.7%). Isolates of B. pertussis were grouped into two groups (G1 and G2) and eight distinct patterns (G1: P1-P5 and G2: P6-P8).CONCLUSION: The resurgence of pertussis should stimulate new research to develop vaccines with greater capacity of protection against current clones and also encourage implementation of new strategies for vaccination in order to reduce the risk of disease in infants.

OBJETIVO: Relatar a incidência, os aspectos epidemiológicos, clínicos, a morte e a vacinação de pacientes com coqueluche e fazer a caracterização genotípica de isolados de Bordetella pertussisidentificados no Estado do Paraná, de janeiro de 2007 a dezembro de 2013.MÉTODOS: Estudo transversal, incluindo 1.209 pacientes com coqueluche. Os dados foram obtidos no Sistema de Informação de Agravos de Notificação (Sinan) e a epidemiologia molecular foi feita por PCR baseada em sequências repetitivas (rep-PCR; DiversiLab(r), bioMerieux, France).RESULTADOS: A incidência de coqueluche no Estado do Paraná aumentou acentuadamente de 0,15-0,76 por 100.000 habitantes entre 2007-2010 para 1,7-4,28 por 100.000 habitantes entre 2011-2013. Os pacientes com menos de um ano foram os mais afetados (67,5%); 59 crianças (5%) desenvolveram coqueluche mesmo depois de receber três doses da vacina e dois reforços com a vacina tríplice DTP. As complicações mais comuns foram pneumonia (14,5%), otite (0,9%) e encefalopatia (0,7%). Isolados de B. pertussis foram agrupados em dois grupos (G1 e G2) e oito padrões distintos (G1: P1-P5 e G2: P6-P8).CONCLUSÃO: O ressurgimento da coqueluche vem para sugerir novas pesquisas com o objetivo se desenvolverem vacinas com maior capacidade de proteção contra os clones atuais e também implantar novas estratégias de vacinação, a fim de reduzir o risco de doenças em lactentes.

Analysis of trinucleotide repetitive sequences for Korean patients with spinocerebellar ataxia types 8, 12, and 17

PURPOSE: Spinocerebellar ataxias (SCAs) are progressive neurodegenerative disorders with diverse modes of inheritance. There are several subtypes of SCAs. SCA 8, SCA 12, and SCA 17 are the less common forms of SCAs with limited information available on their epidemiological profiles in Korea. The purpose of this study was to investigate the prevalence of SCA8, SCA12, and SCA17 in Korea. MATERIALS AND METHODS: Ninety-six unrelated Korean patients were enrolled and showed normal trinucleotide repeats through polymerase-chain reaction (PCR) for the genes ATXN1, ATXN2, ATXN3, CACNA1A , and ATXN7, which correspond to SCA1, SCA2, SCA3, SCA6, and SCA7, respectively. PCR products from patients were further analyzed by capillary electrophoresis using fluorescence labeled primers for the genes ATXN8OS, PPP2R2B, and TBP, which correspond to SCA8, SCA12, and SCA17. RESULTS: Three patients had 104, 97, and 75 abnormal expanded repeats in the ATXN8OS gene, the causative gene for SCA8. None of the patients exhibited abnormal repeats in SCA12 and SCA17. Normal trinucleotide repeat ranges of the cohort in this study were estimated to be 17-34 copies (average, 24+/-4 copies) for SCA8, 7-18 copies (average, 13+/-3 copies) for SCA12, and 26-43 copies (average, 35+/-2 copies) for SCA17. CONCLUSION: This study demonstrated that SCA8, SCA12, and SCA17 are rare in Korean patients with SCA, and further genetic studies are warranted to enhance the mutation detection rate in the Korean SCA population.

Characterization of BZLF1 gene and its promoter Zp of EBV strains in children with EBV-associated diseases in recent 5 years in Beijing area / 病毒学报

This study aims to investigate the genetic characteristics of BZLF1 gene and its promoter Zp of the epidemic strains in children with primary Epstein-Barr virus (EBV)-associated diseases. Total DNA was extracted from the peripheral blood of 134 children with EBV-associated infectious mononucleosis (EBV-IM) and 32 children with EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) who were admitted to Beijing Children's Hospital from 2006 to 2011. The EBNA3C, BZLF1, and Zp genes were amplified by PCR assay. Typing of EBV was performed according to the size of the amplification product of EBNA3C gene; the amplification products of BZLF1 and Zp genes were subjected to direct sequencing, and sequence analysis was performed using BioEdit 7. 0. 9. The results were as follows: (1) EBV-1 was present in 140 samples (97.2%, 140/144) and EBV-II in 4 samples (2.8%, 4/144). (2) Three BZLF1 genotypes and their 12 subtypes (including 6 newly found subtypes) were detected in this study; there were no significant differences in the frequencies of BZLF1-A and BZLF1-B between the children with EBV-IM and EBV-HLH (P = 0.083); BZLF1-A1 was the dominant genotype in children with EBV-associated diseases; t BZLF1-A mostly had three 29-bp repeats in the first intron of BZLF1 gene, and BZLF1-B mostly had 30-bp repeats (P = 0.000), with the number of repeats varying from 1 to 13. (3) Four Zp genotypes were detected in this study, including Zp-P, Zp-V3, Zp-V4, and Zp-V1; there were no significant differences in the frequencies of these Zp genotypes between children with EBV-IM and EBV-HLH (P = 0.272, 0.252, 1.0, and 1.0, respectively). (4) The linkage analysis of BZLF1 gene and its promoter Zp showed that BZLF1-A1 was highly associated with Zp-V3 (P = 0.000), while BZLF1-B4 with Zp-P (P = 0.000); EBV-I + BZLF1 A1 was highly associated with Zp-V3 (P = 0.000), while EBV-I+BZLF1-B4 with Zp-P (P = 0.000). The conclusions are as follows: (1) BZLF1-A1 is the dominant genotype in children with EBV-associated diseases; there are mostly 29-bp repeats in the first intron of BZLF1 gene for BZLF1-A genotype and 30-bp repeats for BZLF1-B genotype. (2) Zp-P and Zp-V3 are dominant Zp genotypes of EBV in children, which shared similar detection rates. (3) BZLF1-A1 is highly associated with Zp-V3, while BZLF1-B4 with Zp-P; EBV-I+BZLF1-A1 is highly associated with Zp-V3, while EBV-I+BZLF1-B4 with Zp-P.

Coxiella burnetii, the agent of Q fever in Brazil: its hidden role in seronegative arthritis and the importance of molecular diagnosis based on the repetitive element IS1111 associated with the transposase gene

Coxiella burnetii is the agent of Q fever , an emergent worldwide zoonosis of wide clinical spectrum. Although C. burnetii infection is typically associated with acute infection, atypical pneumonia and flu-like symptoms, endocarditis, osteoarticular manifestations and severe disease are possible, especially when the patient has a suppressed immune system; however, these severe complications are typically neglected. This study reports the sequencing of the repetitive element IS1111 of the transposase gene of C. burnetii from blood and bronchoalveolar lavage (BAL) samples from a patient with severe pneumonia following methotrexate therapy, resulting in the molecular diagnosis of Q fever in a patient who had been diagnosed with active seronegative polyarthritis two years earlier. To the best of our knowledge, this represents the first documented case of the isolation of C. burnetii DNA from a BAL sample.

Expression of YB-1 enhances production of murine leukemia virus vectors by stabilizing genomic viral RNA

Murine leukemia virus (MLV)-based retroviral vectors is widely used for gene transfer and basic research, and production of high-titer retroviral vectors is very important. Here we report that expression of the Y-box binding protein 1 (YB-1) enhanced the production of infectious MLV vectors. YB-1 specifically increased the stability of viral genomic RNA in virus-producing cells, and thus increasing viral RNA levels in both producer cells and virion particles. The viral element responsive to YB-1 was mapped to the repeat sequence (R region) in MLV genomic RNA. These results identified YB-1 as a MLV mRNA stabilizer, which can be used for improving production of MLV vectors.

Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India

The aim of the present study was to compare polymerase chain reaction (PCR)-based methods - spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing - with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligotype International Types (SITs)26 [Central-Asian (CAS)1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI) 3-Indian lineage]. A large number of isolates (75 percent) belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains) resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.

The Present and Future of Molecular Epidemiology in Tuberculosis

Molecular epidemiology has been initiated for the confirmation of transmission link among tuberculosis patients. IS6110 restriction fragment length polymorphisms (RFLP) technique has been used as an excellent tool to discriminate Mycobacterium tuberculosis isolates, especially in tuberculosis (TB) outbreak in the population. IS6110 RFLP has the most discriminatory power for the M. tuberculosis isolates with high copy number of IS6110 like Korean isolates. Spoligotyping using spacers of direct repeat is useful to distinguish Beijing strains which are found widely in Eastern Asia, from non-Beijing strains. It is known that Beijing strains are more virulent, apt to be drug resistant than non-Beijing strains. Strain typing techniques of Mycobacterium tuberculosis has lead to the development of phylogenetic classification. Variable number tandem repeat (VNTR) of M. tuberculosis is another good target for strain typing. The technique using VNTR is rising as an alternative tool to overcome disadvantages of IS6110 RFLP which is time consuming in the sense that it takes longer time to process from the culture positive bacilli, and has the intrinsic difficulties in objectification of the results. The combination of many VNTR loci enhances discriminatory power to become equal to that of IS6110 RFLP. On the other hand, the optimal VNTR combination differs from one country to another due to different dominant clade. Large sequence polymorphisms (LSP) and single nucleotide polymorphisms (SNP) are important tools for the classification of the phylogeny of M. tuberculosis complex. Many previous reports indicate that the depending upon the type of strains, the ways of transmission of disease, the way to get infected with disease and the development of drug resistance conditions are variable. Therefore, the molecular epidemiology of M. tuberculosis has become more important for tuberculosis control in the world. It will be possible to set up tuberculosis-tailored policy after the characterization of M. tuberculosis by molecular epidemiologically.

Characteristics of Molecular Strain Typing of Mycobacterium tuberculosis Isolated from Korea / 대한임상미생물학회지

Molecular strain typing of Mycobacterium tuberculosis is important for the detection of outbreaks of tuberculosis and laboratory cross contamination, as well as the differentiation between re-infection and reactivation of tuberculosis. In the present review, the authors investigated the currently available typing methods for M. tuberculosis and the current status of strain distribution in Korea. IS6110-restriction fragment length polymorphism (RFLP), which is considered a standard method, is based on numbers and positions of the insertion sequence, IS6110. The method has an excellent discriminatory power with a considerable amount of worldwide data, although it is time-consuming and labor-intensive. Spoligotyping is based on the presence or absence of spacer sequences between direct repeat (DR) regions. PCR amplification allows for the possibility of application in the early suspicious stage. The data can be easily digitized; however, it shows identical profiles in Beijing family strains. Mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) is another PCR-based genotyping method with a good discrimination power whose data can also be easily digitized. In Korea, the prevalence of Beijing family strains have been as high as 80 to 87%.

Study of EST-SSR marker system of Cordyceps / 中国中药杂志

<p><b>OBJECTIVE</b>To establish the EST-SSR marker system for Cordyceps by using ESTs of C. bassiana and C. militaris.</p><p><b>METHOD</b>The ESTs of Cordyceps were downloaded from the public database of NCBI, and the redundant ESTs with low quality were removed. The EST-SSR primers were designed by Sequece Seiner 1. 2. And the primers were screened through PAGE-Electrophoresis.</p><p><b>RESULT</b>The 4 556 non-redundant ESTs which from C. bassiana with total length of 2 953 173 bp were selected. 718 EST-SSRs distributed in 616 ESTs were totally screened out, accounting for 15.8% of the non-redundant ESTs. It was discovered that the average distance of EST-SSSR was 1/4 096 bp in EST-SSRs distribution of C. bassiana. Trinucleotide repeats were the most abundant types with 419 repeated sequences. Regarding to C. militaris, totally 1 363 non-redundant ESTs were acquired, from which 1 117 EST-SSRs were screened, and rate of SSR sites in ESTs was 81.95%. The leading motif of SSR was nucleotide A. The 50 pairs of EST-SSR primers were designed according to the ESTs of C. bassiana, and preliminary test showed the 34 pairs of primers amplified clear fragments,accounting for 68% of all primers. Furthermore, the 39 of the 40 pairs of primers from the ESTs of C. militaris were found to be amplified as the clear fragments, accounting for 97.5%. The phylogenetic analysis revealed that different anamorph of Cordyceps spieces were divided into four branches.</p><p><b>CONCLUSION</b>The EST-SSR of Cordyceps had comparably higher utility value. The EST-SSR markers developed from ESTs of C. bassiana and C. militaris had well transferability in Cordyceps. And it was suggested that the EST-SSR markers should be an easy and effective way to assay molecular genetic structure of Cordyceps.</p>

Researches on relationship between genetic differentiation and chemical variation of Cinnamomum migao / 中国中药杂志

<p><b>OBJECTIVE</b>To study the relationship between the genetic diversity and chemical variation of Cinnamomum migao.</p><p><b>METHOD</b>ISSR marker technique was used to research the genetic structure of 9 population, GC-MS was used to analyze the main ingredients of the volatile oil in C. migao.</p><p><b>RESULT</b>The analysis on the main ingredients of the volatile oil showed that there were significant or extremely significant differences in 9 populations. The minimum variation index of population was Yunnan Funing and the maximum variation index of population was Guangxi Yueye. ISSR marker analysis showed that the average of polymorphic loci percentage (P) was 42.41%, expected heterozygosity (H) was 0.181 0, Shannon's information index (I) was 0.293 8, the Nei's genetic diversity (H(s)) in the group was 0.188 9, genetic differentiation index (G(st)) was 2.269 1. The relationship between the genetic diversity and chemical variation showed that there was no significant correlation between the main ingredients of the volatile oil and 4 indexes of genetic structure of C. migao.</p><p><b>CONCLUSION</b>The genetic diversity of C. migao was relatively high at the population levels, while it is low within the population levels, the relationship between chemical variation and genetic diversity was not obvious, that may indicate that other factors causes the chemical variation of C. migao.</p>