| With the growing number of the patients with AIDS and other immunosuppressive diseases,the incidence of cutaneous mycobacteria infections has been increased in recent years.Because of the nonspecific clinical and pathological presentation,the diagnosis of cutaneous mycobacterial infections mainly depends on detection of the organism. Identification of mycobacteria to species level is crucial since it provides a great deal of useful information on epidemiology and facilitates successful treatment of the patients. Rapid and accurate identification methods are necessary to guide clinicians to treat patients as early as possible.The traditional identification by culture and biochemical tests used to take several weeks.It is difficult for clinicians to acquire valid information immediately according to the conventional procedures.The principal rapid,sensitive and specific methods are cell component analysis and molecular biology methods based on PCR.Among these,PCR-RFLP,a less expensive method,has been proposed for the rapid identification of mycobacteria in clinical laboratory.According to this method,common target sequences of mycobacteria are amplified and digested by one or several kinds of restriction enzymes.Mycobacteria is identified to species by analyzing various numbers and molecular sizes of fragment bands.There are many kinds of common target genes of mycobacteria,such as 16S rRNA, dnaJ,16S-23S rRNA,rpoB and hsp65.PCR-RFLP based on Hsp65 gene need only two incision enzyme to indentify mycobacteria to species.It can identify all kinds of mycobacteria through one test theoretically.Actually some strains must be identified by small fragments and high resolution gel is necessary to analyze.This study is designed to compare the accuracy and sensitivity of the fragment sizes of PCR-RFLP based on Hsp65 gene using 2%agarose gel,2%Metaphor agarose gel and 10%polyacrylamide gel and try to find an ideal high resolution gel to identify mycobacteria in the infectious skin specimen directly.Chapterâ… Compare study of accuracy of fragment sizes of PCR-RFLP based on hsp65 using 2%agarose gel,2%Metaphor agarose gel and 10%o polyacrylamide gel.Ten reference strains were selected.DNA was extracted by'freeze-thawing'method. Hsp65 gene was amplified and digested by BstEâ…¡and Haeâ…¢enzyme.Fragment band sizes were analyzed by 2%agarose gel,2%Metaphor agarose gel and 10% polyacrylamide gel under the same concentration.According to the internet database (http://app.chuv.ch),ten reference strains should be digested to 57 fragment bands.In the experiment,54 fragment bands were acquired by 10%polyacrylamide gel and 3 showed no bands.Compared with the internet database,there were 42 fragment sizes varied within 5 bp,9 within 10 bp,1 within 15 bp and 2 beyond 15 bp.By 2%Metaphor agarose gel 50 fragment bands were acquired and 7 showed no bands.There were 31 fragment sizes varied within 5 bp,10 within 10 bp,8 within 15 bp and 1 beyond 15 bp. By 2%agarose gel 42 fragment bands were acquired and 15 showed no bands.There were 19 fragment sizes varied within 5 bp,13 within 10 bp,9 within 15 bp and 1 beyond 15 bp.Therefore the fragment sizes were closer to the internet database and more accurate in 10%polyacrylamide gel than in 2%agarose gel and 2%Metaphor agarose gel.Chapterâ…¡Compare study of sensitivity of fragment sizes of PCR-RFLP based on hsp65 using 2%agarose gel,2%Metaphor agarose gel and 10%polyacrylamide gel.One M.tuberculosis and one M.intracellulare reference strain,three M.tuberculosis and three M.intracellulare clinical isolates were selected.A series of bacterium suspension were formed.DNA was extracted by'freeze-thawing'method.Hsp65 gene was amplified and digested by BstEâ…¡and Haeâ…¢enzyme.Fragment band sizes were analyzed by 2%agarose gel,2%Metaphor agarose gel and 10%polyacrylamide gel.The sensitivity difference of three methods was analyzed by One-Way ANOVA of SPSS 13.0. In result the fragment sizes of PCP-RFLP were more sensitive in 10%polyacrylamide gel and 2%Metaphor agarose gel than in 2%agarose gel while there was no significant difference in 1.0%polyacrylamide gel and 2%Metaphor agarose gel.Chapterâ…¢Use of PCR-RFLP based on hsp65 to analyze the specificity and sensitivity of clinical specimen suspected of cutaneous mycobacteira infections by 10%polyacrylamide gel.Twenty clinical skin specimens suspected of mycobacteria infection were detected and identified by microscope,culture and PCR-RFLP directly and confirmed by sequencing.It was found that all specimens were negtive under microscope and 10 were positive in culture.Among 10 culture positive specimens,1 was identified to M.arinum and 2 were identified to M.tuberculosis by hsp65 PCR-RFLP. These PCR-RFLP results were agree with the sequencing of hsp65 and 16S rRNA genes. Among 10 culture negative specimens,none was identified by hsp65 PCR-RFLP.It was also found the sensitivity of PCR-RFLP based on hsp65 was 30%(3/10) and specificity 100%(10/10).It might be recoginzed that clinical specimens could be preliminarily indentified by PCR-RFLP based on hsp65 with 10%polyacrylamide gel. |
PDF Full Text Request