Multiplex PCR: rapid DNA cycling in a conventional thermal cycler.

Multiplex polymerase chain reaction (PCR) is a variant of PCR in which two or more target sequences are simultaneously amplified in the same reaction. In the present study we investigated the limits to which the duration of multiplex PCR steps can be shortened using the thermal cycler Gene Amp PCR system 9600 (Perkin Elmer, Oak Brook, IL). The present multiplex PCR assay simultaneously detects five different herpes viruses (HSV-1, HSV-2, VZV, CMV, and EBV) and assesses sample suitability in a single amplification round of 40 cycles. It appears that when six target sequences are simultaneously amplified in multiplex PCR, extension time is a critical parameter. Using a PCR protocol of 0 sec at 95 degrees C, 0 sec at 60 degrees C, and 0 sec at 74 degrees C with Platinum Taq DNA polymerase (Life Technologies, Gaithersburg, MD), we were able to reduce the total cycling time of the multiplex PCR assay to as little as 55 min, without affecting the yield of PCR products or the specificity of the assay. It may be necessary to optimize each specific apparatus and template, but any such optimization would be trivial.

Multiplex polymerase chain reaction: a practical approach.

Considerable time and effort can be saved by simultaneously amplifying multiple sequences in a single reaction, a process referred to as multiplex polymerase chain reaction (PCR). Multiplex PCR requires that primers lead to amplification of unique regions of DNA, both in individual pairs and in combinations of many primers, under a single set of reaction conditions. In addition, methods must be available for the analysis of each individual amplification product from the mixture of all the products. Multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. The development of an efficient multiplex PCR usually requires strategic planning and multiple attempts to optimize reaction conditions. For a successful multiplex PCR assay, the relative concentration of the primers, concentration of the PCR buffer, balance between the magnesium chloride and deoxynucleotide concentrations, cycling temperatures, and amount of template DNA and Taq DNA polymerase are important. An optimal combination of annealing temperature and buffer concentration is essential in multiplex PCR to obtain highly specific amplification products. Magnesium chloride concentration needs only to be proportional to the amount of dNTP, while adjusting primer concentration for each target sequence is also essential. The list of various factors that can influence the reaction is by no means complete. Optimization of the parameters discussed in the present review should provide a practical approach toward resolving the common problems encountered in multiplex PCR (such as spurious amplification products, uneven or no amplification of some target sequences, and difficulties in reproducing some results). Thorough evaluation and validation of new multiplex PCR procedures is essential. The sensitivity and specificity must be thoroughly evaluated using standardized purified nucleic acids. Where available, full use should be made of external and internal quality controls, which must be rigorously applied. As the number of microbial agents detectable by PCR increases, it will become highly desirable for practical purposes to achieve simultaneous detection of multiple agents that cause similar or identical clinical syndromes and/or share similar epidemiological features.

Development of a quadriplex polymerase chain reaction for human cytomegalovirus detection.

The development of a quadriplex PCR method with amplification of HCMV in a single-step procedure using primers taken from four different regions of the viral genome is described. Different concentrations of dNTPs and MgCl2 were assayed in order to optimize the constitution of the buffer for the multiplex PCR. The specificity of the PCR was tested with 100 ng, 10 ng, and 1 ng of genomic MRC-5 cell DNA infected with CMV in the presence of 10 microg of uninfected MRC-5 cell DNA. The sensitivity of the PCR was evaluated by the amplification of various amounts (100 ng, 10 ng, 1 ng, and 0.1 ng) of genomic MRC-5 cell DNA infected with CMV. The specificity and sensitivity assays were performed for each pair of primers and for the combined four primer pairs in the multiplex PCR. CMV was consistently detected from 10 ng of genomic MRC-5 cell DNA with each primer pair. When all four sets of primers were combined in a single reaction tube, the sensitivity of the assay was equivalent to 10 ng of genomic MRC-5 cell DNA, whereas amplification from 1 ng genomic MRC-5 cell DNA produced only a subset of the amplimers. By amplifying four target-sequences of HCMV simultaneously with minimum incubation time at each temperature, a quadriplex, highly sensitive PCR assay was performed. The use of four primer sets designed in different genomic regions of HCMV allowed the detection of variants and achieved maximal sensitivity and specificity which are essential for a diagnostic utilization.

Clear detection and typing of herpes simplex virus types 1 and 2 by an indirect ELISA assay: comparison with three different combined methods--capture ELISA, restriction enzymes, and polymerase chain reaction.

The severity and recurrences of Herpes Simplex Virus (HSV) infection depend on the type of the infectious agent (HSV-1 or HSV-2), which induces the necessity of a nonambiguous detecting typing. The commonly used capture ELISA technique has to be often supported by DNA analysis to confirm the detection and the typing of HSV viruses in exposed patients. In this report, we describe a rapid and cheap indirect ELISA method using anti-HSV monospecific polyclonal antibodies prepared in the laboratory. The typing of the studied samples was clear, did not need series of dilution, and allowed the immediate classification of viruses without further control examination. We tested 51 specimens, which were typed 25 HSV-1 and 26 HSV-2 strains. The comparison with capture ELISA, restriction enzyme and polymerase chain reaction analysis definitely allowed our method to be assessed as a useful tool for a routine diagnostic.

Fast analysis of genomic homologies: primate immunodeficiency virus.

We have recently published a new probabilistic algorithm which performs genomic comparisons on a huge scale. In the present paper it was applied to immunodeficiency viral sequences extracted from international gene databanks. During global sequence analysis of human (HIV1 and HIV2) and simian viruses by means of dot-matrix representation, series of homology were obtained which permitted the definition of families of viruses overlapping the species divisions. Sequences of interest were characterized to the lexical base sentence through successive zoomings. Strain-to-strain comparison confirmed subfamily classifications and led, for example, to the identification of divergent LTR sequences. By way of example, we described the application of the algorithm to the ANT70C and MVP5180 HIV1-O viruses, for which the observed differences were shown to correspond to a deletion in the U3 region, situated between the LEF and NF-kappaB sites. It was of interest to consider these data in a tentative phylogenetic interpretation.

A probabilistic algorithm for interactive huge genome comparison.

We designed a new probabilistic algorithm, named PAGEC (probabilistic algorithm for genome comparison), which allowed a highly interactive study of long genomic strings. The comparison between two nucleic acid sequences is based on the creation of multiple index tables, which drastically reduces processing time for huge genomes, e.g. 13 min for a 4 Mb/4 Mb comparison. PAGEC lowered the need for memory when compared with other types of algorithm and took into account the low resolution of the final representation (paper or computer screen). Considering that standard printers permit a 300 d.p.i. resolution, the loss of computed information due to the probabilistic conception of the algorithm was not usually noticeable in the present study, mainly due to increased genomic sizes. Refinement was possible through an interactive zooming system, which enabled the visualization of the lexical base sequences of a considered part of both of the studied genomes. Biological examples of computation based on yeast and animal nucleic acid sequences presented in this paper reveal the flexibility of the PAGEC program, which is a valuable tool for genetic studies as it offers a solution to an important problem that will become even more important as time passes.

A combined indirect ELISA and immunoblotting for the detection of intrathecal herpes simplex virus IgG antibody synthesis in patients with herpes simplex virus encephalitis.

A combined indirect ELISA and immunoblotting assay was used for the detection of intrathecal synthesis of IgG antibodies to herpes simplex virus (HSV) in patients with HSV encephalitis (HSVE). By using these two assays as well as three markers for blood-brain barrier, leakage can be easily excluded. A total of 21 sera and 24 cerebrospinal fluid (CSF) samples from 11 patients with HSVE were examined. For seven patients more than one pair of serum and CSF were available. For one patient IgG antibodies began to be detectable in CSF after the sixth day from the onset of the disease. In the other 10 patients the intrathecal synthesis of HSV IgG antibodies was detected later than the sixth day and reached high optical density (OD) values after the 10th day from the onset of disease, at the earliest. In contrast, intrathecal HSV antibody synthesis was not found in specimens taken from 20 patients with acute meningitis who composed our negative control group. The use of a combined indirect ELISA and of an immunoblotting assay on a single dilution of serum and CSF for HSV IgG synthesis in the central nervous system (CNS) allowed the diagnosis of HSVE after the first week of disease.

In vitro non-productive infection of purified natural killer cells by the BRU isolate of the human immunodeficiency virus type 1.

Highly purified natural killer (NK) cell lines and clones, displaying the typical phenotype, morphology and function and obtained from healthy blood donors, were infected in vitro with the BRU isolate of human immunodeficiency virus type 1 (HIV-1). There was no significant increase in reverse transcriptase activity and levels of p24 antigen in the supernatants, but positive staining was observed using an immunogold technique with polyclonal anti-HIV-1 antibodies. When infected NK cells were co-cultivated with autologous non-infected CD4+ mitogen-activated cells, significant levels of reverse transcriptase activity and p24 antigen in supernatants were detected. Giant syncytial cells and a high number of mature virion particles were also evident. When NK cell lines or clones from HIV-1-infected patients were studied, neither the presence of p24 antigen nor reverse transcriptase activity was detected in the supernatants after stimulation with mitogens, cytokines or co-culture with allogeneic CD4+ mitogen-activated cells. PCR studies did not detect HIV-1 genes in freshly purified NK cells, cell lines or clones from infected patients. Taken together these results suggest that (i) normal NK cells can be infected in vitro by the HIV-1 BRU isolate in a non-productive fashion, (ii) PCR with NK cell DNA of HIV-1-infected patients indicates that in vivo few of these cells, if any, are infected by HIV-1 and (iii) the mechanisms responsible for the impairment of NK cell function during HIV-1 infection remain to be determined and are probably not related to a direct cytopathic effect of the virus.

Multicentre quality control of polymerase chain reaction for detection of HIV DNA.

OBJECTIVE: Seven French laboratories tested the specificity and sensitivity of the polymerase chain reaction (PCR) for the detection of HIV-1 DNA. METHODS: Following its own PCR protocols, each laboratory independently tested blind two panels of 20 coded peripheral blood mononuclear cell samples collected from HIV-1-seropositive individuals and from HIV-1-seronegative individuals at high or low risk of HIV infection. For the first panel, laboratories were free to select type and number of primers; for the second, all were required to use the two primer pairs Pol 3/4 and MMy 9/10' (Nef 1). RESULTS: False-positive and false-negative results were observed in all laboratories (concordance with serology ranged from 40 to 100%). In addition, the number of positive PCR results did not differ significantly between high- and low-risk seronegatives. The use of crude cell lysates in DNA preparation produced the same PCR results as phenol-extracted DNA. Discrepancies between laboratories indicated that factors other than primer pairs contributed strongly to laboratory variability. CONCLUSIONS: Our results emphasize the importance of both positive and negative controls in PCR and demonstrate the value of multicentre PCR quality control.

Polymerase chain reaction for studies of mother to child transmission of HIV1 in Africa.

The feasibility and implications of the use of the polymerase chain reaction (PCR) assay in studies of HIV1 mother to child transmission in Africa were investigated. Uncultured leukocyte blood cells (PBL) obtained in Brazzaville (Congo) from newborns and infants (mean age = 27 weeks) of infected mothers were tested. HIV1 DNA sequences were identified in the PBL of six of eight newborns and 14 of 23 babies born to HIV1-positive mothers. In addition two of four babies, who at birth had been seropositive and subsequently were seronegative, were HIV1 DNA positive by PCR. This study demonstrates directly, therefore, a high rate of HIV1 transmission in Africa; it also indicates that PCR should be used for such epidemiological studies.

Physical and immunological analysis of the two domains isolated from a variant surface glycoprotein of Trypanosoma brucei.

A specific surface glycoprotein of a variant of Trypanosoma brucei was cleaved with trypsin and the two major domains of the molecule have been purified. We have studied the chemical composition of each domain and compared the data to published results of the specific cDNA sequence. Circular dichroism measurements show that the amino-terminal domain includes preferentially alpha-helical or beta-sheet structure. The physicochemical analyses are supplemented by a prediction of secondary structure and a statistical pattern of hydrophilicity-hydrophobicity. The results are discussed in light of the internal limits that were described in the process of partial gene conversion occurring between the variant gene sequence and related members of the same gene family. Immunoblots with homologous antiserum indicate that the amino-terminal domain is implicated in antigenicity. In addition, immunoblotting with heterologous antiserum on native antigen, tryptic hydrolysates, or purified domains suggests a site of interaction supported by the two domains.

Involvement of the bacterial groM gene product in bacteriophage T7 reproduction. I. Arrest at the level of DNA packaging.

The multiplication of bacteriophage T7 is blocked in Escherichia coli M. The genetic determinant of this ability (groM) to inhibit T7 growth was transferred to an E. coli K-12 recipient by means of conjugation. We determined at which precise step T7 maturation is blocked. Phage-directed protein and DNA synthesis as well as degradation of host DNA were not qualitatively affected. Instead of infective phages, only preheads were produced. These, however, were maturable in vitro. The newly synthesized phage DNA accumulated in a concatemeric form and matured from its tetrameric or longer forms (very fast sedimenting DNA) only into its dimeric form (fast-sedimenting DNA) or longer forms. The following step, i.e., the maturation of the dimeric to unit-length DNA, was not observed. Since the concatemeric form of T7 DNA accumulated in spite of the presence of maturable preheads, it is likely that the maturation process was blocked at the level of DNA packaging. As intermediates in the packaging process, we found some prehead-DNA complexes. We interpreted these as true assembly intermediates (or breakdown products thereof), since the attached DNA was still in its concatemeric form. This shows that the very first DNA packaging step, the binding of the progeny DNA to the preheads, was obviously not blocked. Rather, a later step, such as the filling of the preheads with T7 DNA or the stabilization of completely packaged particles (i.e., the final cutting of the concatemers into unit-size length), was inhibited.

High magnesium content of Escherichia coli B.

We have found that the intracellular concentration of magnesium in exponentially growing Escherichia coli B is much higher than has been previously assumed; it is about 100 mM. Results of equilibrium dialysis suggest that nearly all of this Mg is bound, probably most of it to nucleic acids. These findings could have important consequences for the study of protein-DNA interactions and the in vitro simulation of protein biosynthesis.

Interaction of glucagon and epinephrine in the regulation of adenosine 3',5'-monophosphate-dependent glycogenolysis in the cultured fetal hepatocyte.

The hormonal control of glycogenolysis has been studied in 3-day-cultured fetal rat hepatocytes which contained stored glycogen. A single addition of 10 nM glucagon or 10 nM epinephrine produced an identical maximal glycogenolytic response, which developed within 4 h and ceased thereafter. The amount of glycogen degraded represented 60% of the stored glycogen or 95% of the newly synthesized glycogen after a 4-h preincubation period in the presence of [14C]glucose. The latter result demonstrates that both hormones interact on the same hepatocytes. Stimulation of glycogenolysis by glucagon or or epinephrine was preceded by an accumulation of intracellular cAMP. From the decreasing order of potency of isoproterenol, epinephrine, norepinephrine, and phenylephrine to activate glycogenolysis, it can be concluded that the epinephrine effect is mainly mediated by beta-adrenergic receptors. When glucagon and epinephrine were added simultaneously at maximal concentrations, the glycogenolytic effects were not additive. Moreover, when epinephrine was added 4 h after glucagon, it elicited a second glycogenolytic response, so that the amount of glycogen degraded represented 80% of the stored glycogen. At this stage, a second addition of glucagon was ineffective, and the extent of the glucagon-induced loss of response depended on the size of the first dose of hormone. Cell densensitizatin to glucagon for glycogenolysis was closely related to the associated response in cAMP production. This desensitization was found to be highly specific for glucagon and was accompanied by a defect in specific glucagon binding. The occurrence of a specific negative regulation of the response to glucagon explained how epinephrine was able to mobilize glycogen accumulated in the continued presence of glucagon during hepatocyte development in culture.

Characterization of a new adenovirus type 5 assembly intermediate.

Adenovirus H5 (Ad5) DNA-protein complexes were extracted with ammonium sulphate (0.2 M) from virus-infected HeLa cell nuclei at 18 h after infection. Analysis of the material by centrifugation through discontinuous sucrose gradients in heavy water revealed the existence of several populations of molecules which were identified, in order of increasing buoyant density, as mature DNA-protein complexes, replication complexes, assembly intermediates and virions. When observed under the electron microscope, some of the assembly intermediates showed a capsid with a tail of entirely double-stranded (ds) DNA, or of dsDNA continued by a portion of single-stranded (ss) DNA thickened by a coat of E-72 K DNA binding protein. Singly or doubly-forked Ad5 replicating DNA molecules partially packaged in virus capsids were also observed. It is suggested that these molecules could be assembly intermediates, i.e. one of the first steps of assembly corresponding to virus DNA entering pre-formed capsids or their precursors. The fact that replication was still going on at one end of many of the DNA molecules in the intermediates, while encapsidation was taking place at the other, raises the possibility of a coupled DNA replication-packaging process in the formation of adenovirions.