Standardization and quality control of PCR analyses.

In the very beginning of polymerase chain reaction (PCR) tests entering the field of diagnosis of infectious agents, the introduction of this technology into routine diagnosis was hampered by its frequent tendency to create false-positive results because of contamination. This problem is now widely solved by the introduction of the uracil-N-glycosylase (UNG) anticontamination technology. However, care must still be taken to avoid other sources of producing false positive results. They might additionally derive from human error and/or insufficient PCR amplification and detection protocols. A special case lies in the fact that PCR also amplifies DNA from dead organisms rendering a result diagnostically correct as positive, but clinically as false-positive. In PCR, as in any other diagnostic test, the risk of creating a false-negative result also exists. In such a case, the most probable source besides human error, low target or poor amplification and detection protocols is an inhibition caused by interfering substances in a patient's sample. Strategies to recognize and overcome this issue are discussed in this article. Finally a few results from quality control studies on amplification technologies in the diagnosis of infectious agents are reviewed.

Monitoring treatment of patients with pulmonary tuberculosis: can PCR be applied?

To assess whether PCR is applicable for monitoring the efficacy of antituberculous treatment, respiratory specimens obtained during treatment and follow-up from sputum smear-positive tuberculosis (TB) patients were examined. First, results of smear, culture, and PCR for Mycobacterium tuberculosis complex (MTB) and an internal inhibition control (MCC) were correlated retrospectively on 1,601 respiratory specimens from patients with no previous cultures of MTB. MTB optical density (OD) values increased to a maximum level of 3.5 to 4.0, with both increasing numbers of acid-fast bacilli and CFU. MTB/MCC OD ratios also increased with both smear and culture grading and correlated significantly better with both than the MTB OD value. Second, changes in MTB OD values and MTB/MCC OD ratios were compared with microscopy and culture for MTB in monthly sputa obtained during treatment and follow-up in 22 smear-positive pulmonary TB patients. Declines in MTB/MCC OD ratios during antituberculous treatment and follow-up were observed. Patients with moderate disease reached the baseline after 6 to 8 months of standard antituberculous treatment regimen, whereas patients with extensive disease were predicted to reach the baseline 1 year or more after the initiation of treatment. Although PCR detects both dead and live bacteria, we believe that PCR can be used to assess the efficacy of antituberculous treatment since increases or slow reductions in MTB/MCC OD ratios would indicate nonoptimal treatment, noncompliance, reduced bioavailability of drugs, or resistant strains of MTB and thereby would identify patients at risk for treatment failure or reactivation.

Evaluation of a new biphasic culture system for the recovery of mycobacteria.

A newly developed biphasic culture system (MB-Check) for recovery of mycobacteria was evaluated. The biphasic system consists of a bottle containing selective modified Middlebrook 7H9 broth and a mounted dip slide with chocolate agar and modified Middlebrook 7H11 agar with and without NAP. The system was compared with culture on two egg-based media, Lowenstein medium and a selective Gottsacker medium, using 995 routine specimens and 90 artificially seeded sputa. Mycobacterium tuberculosis was detected in 17 of the 995 routine specimens by the biphasic system and in 14 specimens by the egg-based media together. In the artificially seeded sputa the biphasic system showed higher sensitivity in detection of both tuberculosis complex and non-tuberculous mycobacteria than the egg-based media. The recovery times of the new system were comparable to those of the two conventional culture methods.

Morphological and molecular characterization of several actinophages isolated from soil which lyse Streptomyces cattleya or S. venezuelae.

Several lytic and lysogenic actinophages were isolated from soil samples infected with Streptomyces cattleya and S. venezuelae. The morphologies and some biological properties of the phages, and the physico-chemical characteristics of their DNAs, were compared. Electron micrographs indicated that all the phage heads were of an icosahedral form, but head size and length of the tail varied. Two of the phages had a broad host range; the other isolates could lyse only a limited number of species. The molecular sizes of the phage DNAs were between 32.2 and 98.5 kb as estimated by electron microscopy and restriction enzyme analysis. The same study also indicated that one of the DNA species contained cohesive ends. The G + C content of the DNAs ranged between 45.1 and 74.2 mol % as estimated from melting studies. Sedimentation velocity experiments implied that several of the phage DNAs were probably heavily glycosylated or methylated. These modifications might explain the partial or slow digestion of some of the DNAs by several of the 23 restriction enzymes tested. Protoplasts of the appropriate Streptomyces strains could be efficiently transfected with phage DNA in the presence of 25% (w/v) polyethylene glycol (mol. wt 6000).

Visualization and exact molecular weight determination of a Rhizobium meliloti megaplasmid.

The entire DNA of Rhizobium meliloti MVII /1 cells was isolated preparatively by gentle lysis and sucrose gradient centrifugation. Fractions of the sucrose gradient were investigated by electron microscopy. We found intact megaplasmids in supercoiled and relaxed form and total chromosomes. The length of the megaplasmid could be measured, which allowed an exact determination of the molecular weight. We found a length of 0.48 +/- 0.019 mm equivalent to a molecular weight of about 1000 X 10(6).

RNA polymerase binding sites on the broad host range plasmid RP4.

Binding sites of Escherichia coli RNA polymerase on RP4 plasmid DNA were determined electron microscopically. Comparison of the RNA polymerase binding map and the genetic map of RP4 revealed several strong binding sites outside the well-known RP4 genes. RNA polymerase binding sites for the three antibiotic resistance genes were also detected. Two binding sites were observed for the tra-1 region, whereas the tra-2 and tra-3 regions showed no prominent affinity for RNA polymerase. The genomic regions for the replication origins, oriV (for vegetative replication) and oriT (for transfer replication, equivalent to rlx), both exhibited strong binding to RNA polymerase, as did genomic regions which code for trans-acting replication functions (trfA and trfB).

Molecular characterization of glutamine synthetase from the nitrogen-fixing phototrophic bacterium Rhodopseudomonas palustris.

The phototrophic bacterium Rhodopseudomonas palustris assimilated ammonium via glutamine synthetase and glutamate synthase. Diazotrophic and ammonium-grown cells had high levels of both enzymes, whereas enzymes of alternative assimilatory pathways were absent or had only low activities. Glutamine synthetase was purified to electrophoretic homogeneity within three steps by dye-ligand and ion exchange chromatography. Electron microscopy revealed a dodecameric molecular entity which was in accordance with parameters derived from electrophoretic techniques. The molecular weight of the enzyme monomer was 558000; that of the dodecamer 670000. The amino acid composition of R. palustris glutamine synthetase was determined and compared by a statistical method with other known enzyme compositions from prokaryotic and eukaryotic origins.

ISR1: an insertion element isolated from the soil bacterium Rhizobium lupini.

The insertion element ISR1 was isolated from the soil bacterium R. lupini. In this strain, ISR1 shows a very strong affinity to plasmid RP4. It causes RP4 mutations at the strikingly high frequency of 10(-2) to 10(-1), either by the integration itself or by generating deletions. In E. coli, ISR1 seems to be inactive. No evidence could be obtained for a promoter site on ISR1 or for an ISR1-encoded protein. Our results indicate, however, an ISR1-specific termination signal for either transcription or translation.

Adenine + thymine content of different genes located on the broad host range plasmid RP4.

The genetic map of plasmid RP4 was correlated with its adenine+thymine (AT) map. For this purpose, RP4 DNA was digested with one or both of the restriction enzymes EcoRI and HindIII and the resulting linear RP4 molecules and fragments were partially denatured, examined in the electron microscope and their AT maps were determined using a computer program. From these AT maps the EcoRI and HindIII restriction sites were located on the AT map of RP4. Since the positions of these restriction sites on the genetic map of RP4 are known, the maps could be compared. They revealed a high AT content for the Tn1 transposon and the kanamycin resistance gene. The tra-1 region is also distinguished by a sharply defined AT-rich region, whereas tra-2 and the tetracycline resistance gene have an AT content which is not distinctly different from the average AT content of RP4.

Relationship of group P1 plasmids revealed by heteroduplex experiments: RP1, RP4, R68 and RK2 are identical.

The molecular relationships of the IncP1 plasmids RP1, RP4, R68 and RK2 were tested by electron microscopic examination of heteroduplexes. In several hybridization experiments molecules were detected which had a 7.8% portion of incomplete reannealing. This 'heterologous region' could be explained by the typical renaturation behaviour of the transposon Tn1. The identity of the Tn1 transposon present in RP1 and RP4 was proved by heteroduplex experiments with lambda phage DNA containing this transposon. These results indicated that the plasmids RP1 and RP4 are identical. Additional heteroduplex experiments between plasmids R68.45 and RP8 and between R68.45 and RK2 were performed. R68.45, a derivative of R68, has a small DNA insertion and RP8 can be regarded as a large insertion mutant of RP4; both insertions were used as single-stranded hybridization markers. From the hybrid molecules formed, it was deduced that R68 and RK2 are identical with RP1 and RP4 as far as molecular structure is revealed by the technique used.

Spontaneous degradation of pRD1 DNA into unique size classes is recA dependent.

The his and nif genes of the P1 type plasmid pRD1 were lost at a high frequency in a recA+ but not in a recA- Escherichia coli host during growth in a non-selective medium. 92% of the His- Nif- segregants after 6 subcultures retained the genetic markers of the precursor plasmid RP4, while the remainder lost all of the pRD1 markers with the concomitant loss of ccc-DNA. Plasmids purified from the His- Nif- segregants resembled RP4 in the physical and genetic properties examined. The contour length of pRD1 DNA molecules from a recA- strain was 49 micrometer corresponding to a molecular weight of 101 Mdals and the buoyant density was 1.715 g/cm3. In contrast, the contour lengths of plasmid molecules isolated from a recA+ host carrying pRD1 fell into 3 size classes of 49, 19 and 2 micrometer corresponding to molecular weights of 101, 39 and 4 Mdals respectively and two DNA species of buoyant density 1.715 and 1.719 g/cm3 were observed.

Plasmid replication functions. II. Cloning analysis of the repA replication region of antibiotic resistance plasmid R6-5.

R6-5 is a low copy number, conjugative, FII incompatibility group plasmid that has a molecular length of 102 kb and that specifies resistance against several antibiotics (chloramphenicol, fusidic acid, kanamycin, streptomycin and sulphonamide) and mercury salts. By means of in vitro cloning procedures, mini plasmids have been generated that contain a DNA segment from the essential region of R6-5 that is only 2.6 kb in length. This DNA segment, which consists of two PstI fragments that are adjacent in the parent plasmid, carries all genes and sequences required for the regulated replication and incompatibility properties of R6-5, including its origin of replication, OriV, an essential function that has been designated RepA, and the copy control function, Cop. Three different polypeptides, having monomer molecular weights of 23,000, 10,000 and 9,500 daltons, are synthesized in detectable quantities by minicells carrying pBR322 hybrid plasmids that contain DNA segments from the R6-5 essential region. A spontaneous deletion derivative of a pBR322 hybrid plasmid that carries the R6-5 origin of replication was isolated. Heteroduplex analysis of this derivative plasmid indicates that the deleted DNA segment carries the R6-5 replication origin and that its termini consist of short inverted repeat sequences.

Repetition of tetracycline resistance determinant genes on R plasmid pRSD1 in Escherichia coli.

The 30 megadalton (Mdal)-conjugative, fi- plasmid pRSD1 determines inducible tetracycline resistance (Tc) in Escherichia coli. As shown by restriction analysis, a 3.5 Mdal-EcoRI fragment of pRSD1 spliced into the small plasmid pRSD2124 comprises the entire Tc determinant (tet) region. A restriction map of pRSD1 is presented which includes the location of the tet region and of an "underwound" loop not related to Tc (Burkardt et al., 1978). Selective amplification of tet genes is demonstrated by three lines of evidence. (i) The resistance level of cell harbouring pRSD1 increases approximately tenfold by induction with 10 microgram/ml of tetracycline. Further growth in the presence of 100 microgram/ml of the drug ("tetracycline stress") selects for cells with even higher resistance levels (about 300 microgram/ml) in rec+ cells. In a recA strain, a smaller proportion of cells attains these high resistance levels suggesting the involvement of host recombination. (ii) Electron micrographs of pRSD1-DNA isolated from tetracycline-stressed cells reveal a heterogeneous population of circular DNA molecules ranging between 1.7 and 21.6 micron. The distribution of contour lengths shows a discrete pattern ascribed to the presence of autonomous single- and multiple-copy Tc determinants and to intact plasmids containing zero to six tet regions in tandem repeats. (iii) This interpretation is supported by heteroduplex and restriction analyses which demonstrate the presence of multiple copies of the 3.5 Mdal-element encompassing the tet region in pRSD1 molecules selected by tetracycline stress. It has been concluded that gene amplification leading to tandem repetition of the tet region ensues in pRSD1. Such plasmids confer increased tetracycline resistance and can, thefore, be selected by high doses of the drug.

Fertility inhibition in Rhizobium lupini by the resistance plasmid RP4.

R plasmid RP4 inhibits the fertility of R. lupini. An RP4 carrying R. lupini donor strain is no longer capable of transferring chromosomal genes. After loss of RP4 the R. lupini fertility reappears. Plasmid RP4 spontaneously mutates at high frequency in R. lupini. RP4 mutants which do not inbibit fertility were isolated. These mutants were always transfer-defective, too. It is postulated that the genetic information for fertility inhibition in R. lupini is a substantial part of the transfer unit of the RP4 plasmid.

Electron microscopy and computerized evaluation of some partially denatured group P resistance plasmids.

DNA of the R plasmids RP1, RP4 and RP8 was isolated from various hosts. The lengths of these plasmid molecules were determined by electron microscopy: RP1 and RP4 were about 19 micron long, RP8 measured 31 micron. An RP4 plasmid mutant, designated RP4a, was isolated from Escherichai coli; it was about 1 micron shorter than normal RP4 DNA. To investigate the molecular relationship between RP4, RP4a and RP8 DNAs of these plasmids were partially denatured and examined in the electron microscope. Measurements of the length and denaturation pattern of the DNA molecules were used to construct physical maps. A new computer program was devised for the alignment of the circular molecules, and the effect of variations of different parameters on the reliability of the program was tested. A comparison of the denaturation pattern of RP4 and RP8 indicated that RP8 was composed of total RP4 plus an additional DNA fragment. The RP4a mutant plasmid could be defined as a deletion mutant with loss of 1 micron DNA.

Characterization of a nosocomially significant, multiple drug-resistant strain of Serratia marcescens.

A multiple drug-resistant strain of Serratia marcescens (bacteriocin type 18) was isolated from three clinical patients. The isolates were found to carry a conjugally nontransferable, nonmobilizeable resistance plasmid (R-plasmid) with resistance-(r)-determinants against ten antimicrobial drugs: ampicillin, carbenicillin, chloramphenicol, gentamicin, kanamycin, neomycin, streptomycin, tobramycin, triple sulfonamides, cotrimoxazole, and--possibly--nalidixic acid, as determined with exposure to 'curing' agents (ethidium bromide, acridine orange, and sodium dodecyl sulfate) and by the high rate of spontaneous loss of r-determinants. Dyebuoyant density centrifugation allowed recovery of R-plasmid DNA that measured roughly 24 mum in contour length; after 'curing' with concomitant loss of 9 r-determinants, the contour length of the R-plasmid DNA of one isolate (No. SE 154) had decreased to roughly 15 mum, and none was detected in the sole variant of the isolate that spontaneously had lost 11 r-determinants.