Rapid detection of mycoplasma contamination in cell cultures by enzymatic detection of polymerase chain reaction (PCR) products.

Enzymatic detection of polymerase chain reaction (ED-PCR) was applied for rapid and easy identification of mycoplasmas from contaminated cell culture. This method was based on the capture of amplified products via biotin-streptavidine affinity and the detection of an incorporated hapten in amplified products with enzyme-linked antibody. Primers corresponding to common sequence of Mollicutes in 16S ribosomal RNA dominated gene was used. Nineteen of twenty Mollicutes so far reported as cell contaminants appeared positive by ED-PCR, whereas remaining one, Acholeplasma axanthum, appeared negative. Samples from sixty-two cell culture were tested for contamination of mycoplasmas by means of ED-PCR, cultivation, and electronmicroscopy. The results of ED-PCR were the same as those of cultivating method. The time required for all the detection process in ED-PCR was about 5 hr for 20 samples. We suggest that ED-PCR can be used in the rapid detection of mycoplasms from cell culture.

Relationship between chromosomal alpha-specific suppressors of sir4-11 and polymorphism of the HMRa-bearing fragment.

In the course of studying sir4-11 which is responsible for the non-mating phenotype of asd-homothallism of Saccharomyces cerevisiae, we detected chromosomal alpha-specific suppressors of it. By examining the mating-type cassette constitution of two strains and the spore-clones derived from a diploid culture formed by a mass mating between these strains, we obtained the following results; (1) the HMRa-bearing HindIII-Bg/II restriction fragment was dimorphic, as judged by size, within each tetrad, (2) while one form was common to all tetrads, the other varied among tetrads, (3) spore-clones with the alpha-specific suppressor of sir4-11 had the variant forms while those without had the common form, and (4) while the two parents had the common form, each independent diploid clone had the common form plus a variant form. From these results, we conclude that the mating of cells in certain combinations induces a change of DNA structure at or near HMRa in a mating-pair specific manner and that the change makes HMRa non-derepressible or non-functional when derepressed.

Rapid detection of the mecA gene in methicillin-resistant staphylococci by enzymatic detection of polymerase chain reaction products.

In order to identify methicillin-resistant staphylococci from clinical sources with ease and reliability, enzymatic detection of polymerase chain reaction (ED-PCR) was applied. ED-PCR is based on the capture of amplified products via biotin-streptavidin affinity and the detection of an incorporated hapten in amplified products with an enzyme-linked antibody. In order to identify methicillin-resistant staphylococci of all species, a 150-bp fragment of the mecA gene was targeted for ED-PCR. After PCR was performed with a pair of biotin and dinitrophenol 5'-labeled primers, the reaction mixture was applied to a microtiter well precoated with streptavidin. Thereafter, bound PCR products were detected colorimetrically with alkaline phosphatase-conjugated anti-dinitrophenol antibody. The extraction of DNA from staphylococcal cells for PCR was simplified so that it could be performed within one tube. The total assay, including PCR, took less than 3 h. The sensitivity of mecA gene detection ranged from greater than 5 x 10(2) CFU per tube for Staphylococcus aureus to greater than 5 x 10(3) CFU per tube for Staphylococcus epidermidis. Genotyping results obtained by ED-PCR of 161 tested strains from the colonies (97 strains of S. aureus and 64 strains of coagulase-negative staphylococci) were compared with the phenotypic susceptibilities of the strains to oxacillin. The results of ED-PCR showed excellent agreement with the MICs of oxacillin with very few exceptions; only one strain of S. aureus and two strains of coagulase-negative staphylococci were found to possess the mecA gene, which was discrepant with their phenotypes. Fifty-five blood culture samples were also tested by ED-PCR. For staphylococcal isolates in 33 of the cultures, oxacillin MICs were >4 microgram/ml; 31 of the 33 staphylococcal isolates were determined by ED-PCR to be mecA gene positive. These results suggest that ED-PCR can be used with reasonable confidence in the clinical microbiological laboratory.

Cloning and mapping of the CYS4 gene of Saccharomyces cerevisiae.

A DNA fragment containing the CYS4 gene of Saccharomyces cerevisiae was isolated from a genomic library. The cloned fragment hybridized to the transverse-alternating-field-electrophoresis band corresponding to chromosomes VII and XV. According to the 2 microns DNA chromosome-loss procedure, the cys2 and cys4 mutations, which are linked together and co-operatively confer cysteine dependence, were assigned to chromosome VII. By further mapping involving tetrad analysis, the cys2-cys4 pair was localized between SUP77 (SUP166) and ade3 on the right arm of chromosome VII.

Lateral flagella of vibrios: serological classification and genetical similarity.

Lateral (L-) flagella-having vibrios were classified into 13 H-serogroups (flagellar antigen serogroups) by means of H-agglutination test. Vibrio parahaemolyticus was classified into 3 serogroups, HL1 to 3. V. alginolyticus and V. harveyi were classified into 5 and 3 serogroups, respectively, but 2 of those were serogroups common to the both species. V. fluvialis and V. furnissii constituted a same serogroup, HL8. Cross-reactivity between each serogroup was not observed in H-agglutination test, although some cross-reactivity was observed in gel diffusion test. Furthermore, similarity of DNA sequence of L-flagellar structure gene was demonstrated by dot blot hybridization test with a DNA probe of HL2 L-flagellar gene fragment. These results suggest conservation of DNA sequence of the L-flagellar gene of vibrios.

Preparation of enzyme-conjugated DNA probe and application to the universal probe system.

A general procedure for the cross-linking of enzyme to DNA has been developed for use as a nonradioactive probe. In this method, DNA is transaminated with diaminopropane to introduce primary amino groups into the cytosine residues. Then the amino groups are converted to thiol groups using a heterobifunctional cross-linker. The thiolated DNA is conjugated with the maleimide-introduced enzyme. With this method, alkaline phosphatase was cross-linked to a single-stranded DNA (sspUCRf1). The conjugate was able to detect 5 pg of target DNA (pUCf1 plasmid, 3.2 kbp) fixed onto the nitrocellulose membrane, using a colorimetric assay. The enzyme-conjugated DNA was applied to "the universal probe system," which consisted of two single-stranded DNA probes (a primary probe and a labeled secondary probe). Using alkaline phosphatase-conjugated sspUCRf1 DNA as the secondary probe, the c-myc gene and HBV DNA were detected effectively on Southern and dot-blot hybridization.

Nonradioactive detection of nucleic acid by the universal probe system.

A convenient and nonradioactive method for DNA hybridization tests termed the "Universal probe system" has been developed. This method is based on the principle of sandwich hybridization. This system consists of two single-stranded DNA probes (a primary probe and a biotin-labeled secondary probe). The primary probe is prepared from a chimeric phage-plasmid vector containing the complementary sequence to a target gene. The secondary probe has a sequence complementary to the vector portion of the primary probe and is labeled with biotin via the transamination reaction. An advantage of this method is that the single-stranded primary probe can be prepared with ease by using the chimeric phage-plasmid vector system, thereby avoiding tedious labeling of individually different probes. As the primary probe is not modified with biotin and other labels, it conserves the sequence to be hybridized with a target. Accordingly, the primary probe containing a relatively short hybridizing region (ca. 50 bp) can efficiently hybridize with the target. In fact, the universal probe is sensitive enough to detect a single-copy human gene on Southern blots.

DNA diagnosis of falciparum malaria.

We have developed DNA diagnosis using Universal probe system for the rapid detection of Plasmodium falciparum parasite in blood. We chose the DHFR-TS (dihydrofolate reductase-thymidylate synthase) gene as target for detection which are the junction part (410 bp) of and the DHFR part (790 bp). In the parasite, there is only one copy of target sequence, therefore, the target sequences were amplified by PCR (polymerase chain reaction) to increase the sensitivity. Our hybridization method consists of two probes; a primary probe prepared from a chimeric phage-plasmid vector (pUCf1) containing sequence complementary to the target, and a biotin-labeled secondary probe complementary to a portion of the primary probe, which is detected by the BCIP/NBT method. We showed that the 410 bp was more sensitive than the 790 bp as a target of P. falciparum, and the limit of detection was 10(3) parasites in 1 ml human blood using 410 bp junction part. We also constructed double PCR systems using junction part of DHFR-TS gene. By amplification of the 410 bp of the junction part and reamplification of 228 bp of inside sequence of the 410 bp, as little as 10 parasites in 10 microliters human blood was sufficient for specific detection.

Applications of universal probe on DNA hybridization.

A convenient method for DNA hybridization termed "Universal probe" is described which is based on the principle of sandwich hybridization. This system consists of two probes: primary probe which is single-stranded DNA prepared from a chimeric phage-plasmid vector containing the complementary sequence to a target; and labeled secondary probe which has an opposite strand of the primary probe without the complementary sequence. By use this universal probe human beta-globin gene was able to be detected on Southern blots of genomic DNA. A potential advantage of this method is that the single-stranded primary probe is prepared easily by the chimeric phage-plasmid vector system and tedious labeling is not needed each time.

[Chronic toxicity test of garlic extract in rats].

The influence of garlic extract on the chronic toxicity test were examined orally in Wistar rats for 6 months. There were no toxic symptoms due to garlic extract even at dose level of 2000 mg/kg for 5 times a week during 6 months. High dose of garlic extract did not inhibit the body weight gain, while the food consumption decreased slightly for the nutritional effects of it in both male and female rats. There were no significant differences in urinary, hematological and serological examinations compared each groups. In the histopathological findings, no toxic signs were observed on any of the tissues and organs examined.