PCR template preparation for capillary DNA sequencing.

Fluorescence-based capillary DNA sequencing has facilitated the early completion of several complex sequencing projects. While capillary systems offer great benefits in terms of ease of use and automation, we find that they are sufficiently different from slab gel separation methodologies, demanding re-examination of the protocols used to generate and use DNA sequencing templates. We have recently initiated a large-scale Human Open Reading Frame EST project involving 30 laboratories feeding 11 MegaBace 1000 capillary sequencers. The group has already produced more than 300,000 valid sequences. The most successful template preparation protocol we have found is described here. We have found that a crucial step is the standardization of the quantity and quality of the templates, which have been achieved by overnight bacterial culture followed by PCR using limiting amounts of primers. Using this protocol, there is no need for post-PCR purification, and the final preparation cost is US $0.09/template. After sequencing 10,848 templates using this protocol, 78% of the reads were accepted (after discarding vectors without inserts and inserts smaller than 100 nucleotides), and 85% of the total number of bases had Phred scores of 15 or above.

Systemic mycobacterioses in AIDS patients as determined by blood cultures on biphasic medium.

Bacteremia due to mycobacteria can occur in AIDS patients in whom a rapid diagnosis is extremely important in order to plan a therapeutic conduct. Blood culture of mycobacteria using a biphasic system was set up in the Regional Laboratories of the Adolfo Lutz Institute, SP (Campinas, Ribeirão Preto, Santo André, Santos, São José do Rio Preto and Sorocaba). During a three year period (1994-97), 1521 blood samples were analyzed from 1336 AIDS patients, with CD4+ cell count < 100/ml, hematocrit < 30% and serum albumin concentration < 3.0 g/dl seen in regional outpatient clinics or as inpatients in hospitals. Of the blood samples examined, 9.9% were positive for mycobacteria. The predominant species was Mycobacterium avium complex (MAC) (53.8%) followed by Mycobacterium tuberculosis (28.0%). Mycobacterium xenopi was isolated in one case (0.8%) and in the remaining 17.4% the mycobacteria isolated were not identified. The implementation of blood culture for mycobacteria in our Institute has permitted the laboratory diagnosis of mycobacterial infections, in addition to providing data on the frequency of disseminated mycobacterial disease in AIDS patients in the region.

Systemic mycobacterioses in AIDS patients as determined by blood cultures on biphasic medium

Bacteremia due to mycobacteria can occur in AIDS patients in whom a rapid diagnosis is extremely important in order to plan a therapeutic conduct. Blood culture of mycobacteria using a biphasic system was set up in the Regional Laboratories of the Adolfo Lutz Institute, SP (Campinas, RibeirÒo Preto, Santo AndrÚ, Santos, SÒo JosÚ do Rio Preto and Sorocaba). During a three year period (1994-97), 1521 blood samples were analyzed from 1336 AIDS patients, with CD4+ cell count < 100/ml, hematocrit < 30 and serum albumin concentration < 3.0 g/dl seen in regional outpatient clinics or as inpatients in hospitals. Of the blood samples examined, 9.9 were positive for mycobacteria. The predominant species was Mycobacterium avium complex (MAC) (53.8) followed by Mycobacterium tuberculosis (28.0). Mycobacterium xenopi was isolated in one case (0.8) and in the remaining 17.4 the mycobacteria isolated were not identified. The implementation of blood culture for mycobacteria in our Institute has permitted the laboratory diagnosis of mycobacterial infections, in addition to providing data on the frequency of disseminated mycobacterial disease in AIDS patients in the region.

Systemic mycobacterioses in AIDS patients as determined by blood cultures on biphasic medium

Bacteremia due to mycobacteria can occur in AIDS patients in whom a rapid diagnosis is extremely important in order to plan a therapeutic conduct. Blood culture of mycobacteria using a biphasic system was set up in the Regional Laboratories of the Adolfo Lutz Institute, SP (Campinas, RibeirOo Preto, Santo AndrU, Santos, SOo JosU do Rio Preto and Sorocaba). During a three year period (1994-97), 1521 blood samples were analyzed from 1336 AIDS patients, with CD4+ cell count < 100/ml, hematocrit < 30 and serum albumin concentration < 3.0 g/dl seen in regional outpatient clinics or as inpatients in hospitals. Of the blood samples examined, 9.9 were positive for mycobacteria. The predominant species was Mycobacterium avium complex (MAC) (53.8) followed by Mycobacterium tuberculosis (28.0). Mycobacterium xenopi was isolated in one case (0.8) and in the remaining 17.4 the mycobacteria isolated were not identified. The implementation of blood culture for mycobacteria in our Institute has permitted the laboratory diagnosis of mycobacterial infections, in addition to providing data on the frequency of disseminated mycobacterial disease in AIDS patients in the region.(AU)

Systemic mycobacterioses in AIDS patients as determined by blood cultures on biphasic medium.

Bacteremia due to mycobacteria can occur in AIDS patients in whom a rapid diagnosis is extremely important in order to plan a therapeutic conduct. Blood culture of mycobacteria using a biphasic system was set up in the Regional Laboratories of the Adolfo Lutz Institute, SP (Campinas, RibeirÒo Preto, Santo André, Santos, SÒo José do Rio Preto and Sorocaba). During a three year period (1994-97), 1521 blood samples were analyzed from 1336 AIDS patients, with CD4+ cell count < 100/ml, hematocrit < 30

and serum albumin concentration < 3.0 g/dl seen in regional outpatient clinics or as inpatients in hospitals. Of the blood samples examined, 9.9

were positive for mycobacteria. The predominant species was Mycobacterium avium complex (MAC) (53.8

) followed by Mycobacterium tuberculosis (28.0

). Mycobacterium xenopi was isolated in one case (0.8

) and in the remaining 17.4

the mycobacteria isolated were not identified. The implementation of blood culture for mycobacteria in our Institute has permitted the laboratory diagnosis of mycobacterial infections, in addition to providing data on the frequency of disseminated mycobacterial disease in AIDS patients in the region.