DETECTION OF HUMAN PAPILLOMAVIRUS TYPES 16, 18 DNA RELATED SEQUENCES IN BRONCHOGENIC CARCINOMA BY POLYMERASE CHAIN REACTION

In studying the relationship between human papillomavirus (HPV) and bronchogenic carcinoma, 'high-risk' HPV 16, 18 DNA sequences were detected in samples from 50 lung cancer patients, 18 patients with benign pulmonary diseases and 4 fetal lung tissues by polymerase chain reaction (PCR) and dot-blot hybridization with biotin-labelled probes. The results showed that HPV 16, 18 DNA related sequences were found in 32% of lung cancer specimens, with 10 cases of HPV 16, 5 cases of HPV 18 and 1 case of both types. 48.15% (13 / 27) of squamous cell carcinomas were shown to be positive for HPV 16, 18 DNA. In addition, two adenocarcinomas and one small cell carcinoma were positive for HPV 16 DNA. No specimens from benign diseases tissues and fetal lung tissues showed positive results. These results suggest that primary bronchogenic carcinoma is related to HPV infection.

Sequence Analysis of Attachment Gene of Lumpy Skin Disease and Sheep Poxviruses

In Egypt,protection of cattle against lumpy skin disease (LSD) was carried out using a sheep poxvirus (Kenyan strain) vaccination strategy.In the present study 15 skin nodules from LSD suspected cows and 5 scab samples from sheep pox (SP) suspected sheep were collected.Hyperimmune rabbit sera to Lumpy skin disease virus (LSDV)/Ismailyia88 strain and sheep pox virus (SPV)/ Kenyan vaccinal strain were prepared.The causative agent in the collected samples was identified using immunoflourescence (IF) and immunoperoxidase techniques.Of the 15 skin nodules suspected of LSD,10 showed a positive reaction and 3 out of 5 skin scabs suspected of sheeppox were found to be positive.An antigenic correlation between field skin isolate of LSDV,tissue culture adapted LSDV/Ismailyia88 strain,field skin isolate of SPV and SPV/Kenyan vaccinal strain was studied using prepared hyperimmune sera.Also,nucleotide sequence of the PCR amplified attachment gene fragments of field skin isolate of LSDV,tissue culture adapted LSDV/Ismailyia88 strain,field skin isolate of SPV and SPV /Kenyan vaccinal strain were compared.The results revealed that the four used viruses were antigenically identical.Sequence analysis indicated that field skin LSDV isolate is more related to tissue culture adapted LSDV/Ismailyia88 strain than to vaccinal SPV/ Kenyan strain and the skin isolate of SPV is more closely related to field skin isolate of LSDV than to SPV/Kenyan vaccinal strain.Thus,further study should be applied on the advantage of a LSD vaccine prepared from LSDV in protection of cattle against LSD compared to the commonly used sheep pox vaccine.

Detection of mutation in embB gene of Mycobacterium tuberculosis from clinical isolates of tuberculous patients in China by means of reverse-dot blot hybridization

The relationship between embB mutation of Mycobacterium tuberculosis and ethambutol （EMB） resistance of the clinical isolates of tuberculous patients in China was investigated by reversedot blot hybridization （RDBH） in addition to evaluating the clinical value with application of PCR-RDBH technique to detect EMB resistance. In the present study, the genotypes of the 258 bp fragments of embB genes from 196 clinical isolates of M. tuberculosis were analysed with RDBH and DNA sequencing. It was demonstrated that 60 out of 91 phenotypically EMB-resistant isolates （65.9%） showed 5 types of missense mutations at codon 306 of embB gene, resulting in the replacement of the Met residue of the wild type strain with Val, Ile or Leu residues. In these mutations, the GTP mutation （38/91, 41.8% ） and the ATA mutation （16/91, 17.6% ） were the most encountered genotypes. The embB mutation at codon 306 could also be found in 69 isolates of phenotypically EMB-sensitive but resistant to other anti-tuberculous drugs, but no such gene mutation could be found in 36 strains of drug-sensitive isolates. Meanwhile, the concordance with the results of DNA sequencing fcr one wide-type probe and 5 probes for specific mutations was 100%. It was concluded that the EMB-resistance occurring in most M. tuberculosis is due to appearance of embB mutation at codon 306, and the PCR-RDBH assay was proved to be a rapid, simple and reliable method for the detection of gene mutations, which might be a good alternative for the drug-resistance screening.

Point mutations of muscle mitochondrial DNA from patients with mitochondrial encephalomyopathies

Objective To study the relation between point mutations at nt3243 and nt8344 of muscle mitochondrial DNA from patients with mitochondrial encephalomyopathies and phenotypes. Methods DNA was extracted from muscle specimens from 5 patients with mitochondrial encephalomyopathies and amplified by PCR method, using corresponding oligonucleotide primers. DNA fragments were digested with restriction enzymes BglⅠ and ApaⅠ, then the digested DNA fragments were analyzed with an electrophoresis method.Results The point mutation at nt3243 of mtDNA was found in 2 patients, one with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) and another with myoclonic epilepsy with ragged red fibers (MERRF). The point mutation at nt8344 was found in 2 patients with MERRF, including the one with point mutation at nt3243.Conclusion The point mutation of DNA at nt3243 correlated with MELAS and nt8344 correlated with MERRF. In addition, the detection of point mutations at both nt3243 and nt8344 in a patient with MERRF shows the association of mutation with diversity in clinical manifestations of mitochondrial encephalomyopathies.

Difference in DNA sequences in SSU rDNA variable regions among pathogens isolated from different epidemic foci of visceral leishmaniasis in China

To confirm the existence of point mutations in the SSU rDNA variable regions of 5 Leishmania donovani (L d ) isolates from different epidemic foci in China Methods Specific SSU rDNA fragments from nuclear DNA of 7 Leishmania species/isolates were amplified by PCR and then cloned into pGEM R T Easy Vectors After that, the specific fragments were sequenced by an automated DNA sequencer Results Sequence analysis showed that the amplified DNA fragments of 7 Leishmania species/isolates were all 392 bp in length All 5 point mutations were located in two unique sequence blocks (UQ Ⅰ and UQ Ⅱ), and no insertions or deletions were found The identities of comparison of Leishmania in GeneBank were more than 98% Conclusion Five point mutations exist in the SSU rDNA variable region of 5 L d isolates from different epidemic foci of visceral leishmaniasis (VL) in China Sequence differences of the SSU rDNA variable region exist among L d isolates from different foci

Study on vertical transmision of Chlamydia trachomatis using PCR and DNA sequencing

Objective To investigate the vertical transmission rate of Chlamy dia trachomatis (CT) in Chongqing, China. Methods Specimens taken from 278 women and from their 79 infants were e xamined b y cell culture, polymerase chain reaction (PCR) and DNA sequence analysis. Chlam ydia trachomatis was isolated in McCoy cell culture. CT DNA was extracted with a modified NaI method. After cloning, recombinant plasmids were used for sequence analysis with the dideoxy chain termination method.Results 10.8% (30/278) of the cervical cultures of pregnant women were positive for Chlamydia trachomatis, while the positive rate tested by PCR was 14.0% (39/2 78). The vertical transmission rate of Chlamydia trachomatis was 55. 0% (11/20). The incidences of conjunctivitis and pneumonia in infants with Chlamydia t rachom atis positive mothers were 27.3% and 18.2%, respectively. DNA sequence s of Chlam ydia trachomatis isolated from the cervix of a mother and the nasopharynx of her baby were identical.Conclusion Chlamydia trachomatis infection is quite common in Cho ngqing , China. Our report is the first report of CT vertical transmission proved by DN A sequence analysis.