Usefulness of a Nested-polymerase chain reaction for molecular diagnosis of human T-cell lymphotropic virus type I/II

This study aimed at implementing a Nested-polymerase chain reaction (Nested-PCR) for the molecular diagnosis of human T-cell lymphotropic virus type I/II (HTLV-I and HTLV-II) infections in peripheral blood mononuclear cells of infected subjects in Argentina. The sensitivity and specificity of the assay for the detection of regional strains were assessed by comparing them with the molecular assay of reference PCR-hybridization. The Nested-PCR detected 1 MT-2 cell (> or = 8 proviral copies)/1x10(6) non-infected cells showing high sensitivity for provirus detection. While both molecular assays showed high specificity (100%) for HTLV-I and HTLV-II detection, the sensitivity values differed: 100% for Nested-PCR and 67% for PCR-hybridization assay. Moreover, this technique showed less sensitivity for the detection of DNA sequences of HTLV-II (33%) than for the detection of DNA sequences of HTLV-I (75%). The high sensitivity and specificity of the Nested-PCR for regional strains and its low costs indicate that this assay could replace the PCR-hybridization assay for the molecular diagnosis of HTLV-I/II infections. It will be interesting to assess the usefulness of this assay as a tool for the molecular diagnosis of HTLV-I/II infections in other developing countries. Other studies that include a greater number of samples should be conducted.     

Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis

In the attempt to explore complex bacterial communities of environmental samples, primers hybridizing to phylogenetically highly conserved regions of 16S rRNA genes are widely used, but differential amplification is a recognized problem. The biases associated with preferential amplification of multitemplate PCR were investigated using 'universal' bacteria-specific primers, focusing on the effect of primer mismatch, annealing temperature and PCR cycle number. The distortion of the template-to-product ratio was measured using predefined template mixtures and environmental samples by terminal restriction fragment length polymorphism analysis. When a 1 : 1 genomic DNA template mixture of two strains was used, primer mismatches inherent in the 63F primer presented a serious bias, showing preferential amplification of the template containing the perfectly matching sequence. The extent of the preferential amplification showed an almost exponential relation with increasing annealing temperature from 47 to 61 degrees C. No negative effect of the various annealing temperatures was observed with the 27F primer, with no mismatches with the target sequences. The number of PCR cycles had little influence on the template-to-product ratios. As a result of additional tests on environmental samples, the use of a low annealing temperature is recommended in order to significantly reduce preferential amplification while maintaining the specificity of PCR.     

The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction

The polymerase chain reaction (PCR) is most effectively performed using a thermostable DNA polymerase such as that isolated from Thermus aquaticus. Since temperature and oligonucleotide length are known to control the specificity of oligonucleotide hybridization, we have investigated the effect of oligonucleotide length, base composition, and the annealing temperature on the specificity and efficiency of amplification by the PCR. Generally, the specificity of PCR is controlled by the length of the oligonucleotide and/or the temperature of annealing of the primer to the template. An empirical relationship between oligonucleotide length and ability to support amplification was determined. This relationship allows for the design of specific oligonucleotide primers. A model is proposed which helps explain the observed dependence of PCR on annealing temperature and length of the primer.     

Preliminary development of a diagnostic test for Brucella using polymerase chain reaction

A highly sensitive and specific diagnostic test for Brucella based on polymerase chain reaction is under development in our laboratory. A commercially available PCR kit was used to create primers that allowed the amplification of a 635 bp fragment of a 43 kDa outer membrane protein gene from Brucella abortus strain 19. We successfully amplified the cloned gene present in the pMS64 plasmid and genomic Brucella S19 DNA. The amplified DNA was easily detected by agarose gel electrophoresis. Using both the pMS64 plasmid and Br. abortus S19 purified DNA as template each component of the PCR reaction was adjusted for the optimum amplification of the DNA sequence. Optimum specific amplification resulted when the primer annealing temperature was 60C. The gene fragment was amplifiable in 25 different Brucella species and strains. To test the specificity of the reaction, DNA extracted from 17 micro-organisms possibly associated with cattle were tested. No amplification was observed. The sensitivity of the reaction was determined with different concentrations of genomic Brucella strain 19 DNA. As little as 0.1 pg DNA (less than 100 brucella cells) could be detected. The specificity and sensitivity of PCR combined with its simplicity and speed suggests the potential of this technique for routine diagnosis of brucellosis.     

Preliminary development of a diagnostic test for Brucella using polymerase chain reaction

A highly sensitive and specific diagnostic test for Brucella based on polymerase chain reaction is under development in our laboratory. A commercially available PCR kit was used to create primers that allowed the amplification of a 635 bp fragment of a 43 kDa outer membrane protein gene from Brucella abortus strain 19. We successfully amplified the cloned gene present in the pMS64 plasmid and genomic Brucella S19 DNA. The amplified DNA was easily detected by agarose gel electrophoresis. Using both the pMS64 plasmid and Br. abortus S19 purified DNA as template each component of the PCR reaction was adjusted for the optimum amplification of the DNA sequence. Optimum specific amplification resulted when the primer annealing temperature was 60 degrees C. The gene fragment was amplifiable in 25 different Brucella species and strains. To test the specificity of the reaction, DNA extracted from 17 micro-organisms possibly associated with cattle were tested. No amplification was observed. The sensitivity of the reaction was determined with different concentrations of genomic Brucella strain 19 DNA. As little as 0.1 pg DNA (less than 100 brucella cells) could be detected. The specificity and sensitivity of PCR combined with its simplicity and speed suggests the potential of this technique for routine diagnosis of brucellosis.     

多倍体罗汉果PCR-RFLP参数的优化与应用

以多倍体罗汉果DNA为材料,采用L16(4~5)正交组合试验和单因素梯度试验,研究Mg~(2+)、dNTP、引物、Taq DNA聚合酶、模板DNA浓度和退火温度、循环次数等对PCR扩增结果以及内切酶量、酶切时间对酶切反应的影响。结果表明,多倍体罗汉果RFLP最优PCR反应体系和扩增参数为:在25μL扩增反应体系中,10×Buffer 2.5μL,MgCl_2 1.5 mmol/L,dNTP 0.2 mmol/L,引物0.1μmol/L,Taq DNA聚合酶2.0 U,模板DNA 60 ng;退火温度为56℃,循环次数为35次。酶切反应体系:内切酶10×Buffer 2.0μL,内切酶5.0U,PCR产物15μL,超纯水补至20μL;酶切时间2 h。     

Recent advances in detection of human T-cell leukemia viruses type I and type II infection

The human T-cell leukemia viruses type I (HTLV-I) and type II (HTLV-II) have been implicated in the pathogenesis of a variety of neoplastic and neurological disorders. Classical techniques for detection involve assay of serum for antibodies by Western blotting or ELISA, which do not discriminate between infection with HTLV-I and HTLV-II. In order to provide appropriate prognostic information to infected individuals and to obtain an accurate assessment of the prevalence of both retroviruses in the United States, we and others have applied the technique of enzymatic DNA amplification to detect HTLV-I and HTLV-II. These techniques allow rapid detection of viral nucleic acids in freshly isolated peripheral blood samples. Recent studies indicate an unusually high rate of HTLV-II infection among seropositive individuals in a sampling of New Orleans intravenous drug users, indicating a need for combined serological and molecular genetic screening of high-risk populations.     

Genetic heterogeneity in human T-cell leukemia/lymphoma virus type II.

DNA from the peripheral blood mononuclear cells of 17 different individuals infected with human T-cell lymphoma/leukemia virus type II (HTLV-II) was successfully amplified by the polymerase chain reaction (PCR) with the primer pair SK110/SK111. This primer pair is conserved among the pol genes of all primate T-cell lymphoma viruses (PTLV) and flanks a 140-bp fragment of DNA which, when used in comparative analyses, reflects the relative degree of diversity among PTLV genomes. Cloning, sequencing, and phylogenetic comparisons of these amplified 140-bp pol fragments indicated that there are at least two distinct genetic substrains of HTLV-II in the Western Hemisphere. These data were confirmed for selected isolates by performing PCR, cloning, and sequencing with to 10 additional primer pair-probe sets specific for different regions throughout the PTLV genome. HTLV-II isolates from Seminole, Guaymi, and Tobas Indians belong in the new substrain of HTLV-II, while the prototype MoT isolate defines the original substrain. There was greater diversity among HTLV-II New World strains than among HTLV-I New World strains. In fact, the heterogeneity among HTLV-II strains from the Western Hemisphere was similar to that observed in HTLV-I and simian T-cell lymphoma/leukemia virus type I isolates from around the world, including Japan, Africa, and Papua New Guinea. Given these geographic and anthropological considerations and assuming similar mutation rates and selective forces among the PTLV, these data suggest either that HTLV-II has existed for a long time in the indigenous Amerindian population or that HTLV-II isolates introduced into the New World were more heterogeneous than the HTLV-I strains introduced into the New World.     

Construction of tandem repeats of DNA fragments by a polymerase chain reaction-based method

We describe a new application of megaprimer polymerase chain reaction (PCR) for constructing a tandemly repeated DNA sequence using the drought responsive element (DRE) from Arabidopsis thaliana as an example. The key feature in the procedure was PCR primers with partial complementarity but differing melting temperatures (T(m)). The reverse primer had a higher T(m), a 3' end complementary to the DRE sequence and a 5' region complementary to the forward primer. The initial cycles of the PCR were conducted at a lower primer annealing temperature to generate products that served as megaprimers in the later cycles conducted at a higher temperature to prevent annealing of the forward primer. The region of overlap between the megaprimers was extended for generating products with a variable copy number (one to four copies) of tandem DRE sequence repeats (71?bp). The PCR product with four tandem repeats (4× DRE) was used as a template to generate tandem repeats with higher copies (copy number large than four) or demonstrated to bind DRE-binding protein in an yeast one-hybrid assay using promotorless reporter genes (HIS and lacZ). This PCR protocol has numerous applications for generating DNA fragments of repeated sequences.     

葡萄ISSR-PCR反应体系的建立与优化

采用正交设计L9(34)对影响葡萄ISSR-PCR反应体系的4个因素(dNTP、TaqDNA聚合酶、引物、模板DNA)在3个浓度水平上进行试验,并通过直观分析初步确定其反应体系;在此基础上,通过单因素试验探讨了dNTP、TaqDNA聚合酶、引物、模板DNA、退火温度及循环次数等因素或条件对葡萄ISSR-PCR扩增结果的影响,确定最佳反应水平。最终建立了葡萄ISSR-PCR扩增的最佳反应体系:在25μL的反应体系中,dNTP浓度0.2 mmol/L,TaqDNA聚合酶的用量0.5 U,引物浓度0.4mmol/L,DNA模板用量40 ng。反应程序:94℃预变性5 min;94℃变性1 min,52℃退火1 min,72℃延伸1 min 30 s,40次循环;最后72℃延伸10 min,10℃保存。     

Locked nucleic acid inhibits amplification of contaminating DNA in real-time PCR

Locked nucleic acid (LNA) is a modified DNA with increased binding affinityfor complementary DNA sequences. Our strategy was to use this property of LNA to inhibit undesired PCR amplification (e.g.,from contaminating genomic DNA) in a cDNA-based assay. By placing a short complementary LNA sequence in intronic DNA, the aim was to inhibit the amplification of genomic DNA without affecting the amplification of reverse-transcribed spliced mRNA. LNA was designed to bind within intron 5 in the x-box binding protein 1 (XBP1) gene. An irrelevant LNA oligonucleotide served as a negative control. In both PCR and real-time PCR, the addition of LNA showed blocking of the amplification of genomic XBP1 but not cDNA XBP1. To test the effect of melting temperature (Tm) on the LNA, we investigated the number of LNA nucleotides that could be replaced with DNA nucleotides and still retain the blocking activity. More than three DNA nucleotides reduced the LNA inhibition ability. The sequence specificity of the LNA was tested by investigating the number of LNA nucleotide mismatches permitted. The introduction of one mismatch maintained the inhibition of genomic amplification whereas two mismatches reduced the amplification. Our results show that LNA may be used to enhance the specificity of PCR by eliminating unwanted PCR products.     

Selective and sensitive method for PCR amplification of Escherichia coli 16S rRNA genes in soil

A set of PCR primers targeting 16S rRNA gene sequences was designed, and PCR parameters were optimized to develop a robust and reliable protocol for selective amplification of Escherichia coli 16S rRNA genes. The method was capable of discriminating E. coli from other enteric bacteria, including its closest relative, Shigella. Selective amplification of E. coli occurred only when the annealing temperature in the PCR was elevated to 72 degrees C, which is 10 degrees C higher than the optimum for the primers. Sensitivity was retained by modifying the length of steps in the PCR, by increasing the number of cycles, and most importantly by optimizing the MgCl(2) concentration. The PCR protocol developed can be completed in less then 2 h and, by using Southern hybridization, has a detection limit of ca. 10 genomic equivalents per reaction. The method was demonstrated to be effective for detecting E. coli DNA in heterogeneous DNA samples, such as those extracted from soil.     

Detection of coliform bacteria in water by polymerase chain reaction and gene probes

Polymerase chain reaction (PCR) amplification and gene probe detection of regions of two genes, lacZ and lamB, were tested for their abilities to detect coliform bacteria. Amplification of a segment of the coding region of Escherichia coli lacZ by using a PCR primer annealing temperature of 50 degrees C detected E. coli and other coliform bacteria (including Shigella spp.) but not Salmonella spp. and noncoliform bacteria. Amplification of a region of E. coli lamB by using a primer annealing temperature of 50 degrees C selectively detected E. coli and Salmonella and Shigella spp. PCR amplification and radiolabeled gene probes detected as little as 1 to 10 fg of genomic E. coli DNA and as a few as 1 to 5 viable E. coli cells in 100 ml of water. PCR amplification of lacZ and lamB provides a basis for a method to detect indicators of fecal contamination of water, and amplification of lamB in particular permits detection of E. coli and enteric pathogens (Salmonella and Shigella spp.) with the necessary specificity and sensitivity for monitoring the bacteriological quality of water so as to ensure the safety of water supplies.     

Detection of coliform bacteria in water by polymerase chain reaction and gene probes.

Polymerase chain reaction (PCR) amplification and gene probe detection of regions of two genes, lacZ and lamB, were tested for their abilities to detect coliform bacteria. Amplification of a segment of the coding region of Escherichia coli lacZ by using a PCR primer annealing temperature of 50 degrees C detected E. coli and other coliform bacteria (including Shigella spp.) but not Salmonella spp. and noncoliform bacteria. Amplification of a region of E. coli lamB by using a primer annealing temperature of 50 degrees C selectively detected E. coli and Salmonella and Shigella spp. PCR amplification and radiolabeled gene probes detected as little as 1 to 10 fg of genomic E. coli DNA and as a few as 1 to 5 viable E. coli cells in 100 ml of water. PCR amplification of lacZ and lamB provides a basis for a method to detect indicators of fecal contamination of water, and amplification of lamB in particular permits detection of E. coli and enteric pathogens (Salmonella and Shigella spp.) with the necessary specificity and sensitivity for monitoring the bacteriological quality of water so as to ensure the safety of water supplies.     

Bovine serum albumin further enhances the effects of organic solvents on increased yield of polymerase chain reaction of GC-rich templates

ABSTRACT: BACKGROUND: While being a standard powerful molecular biology technique, applications of the PCR to the amplification of high GC-rich DNA samples still present challenges which include limited yield and poor specificity of the reaction. Organic solvents, including DMSO and formamide, have been often employed as additives to increase the efficiency of amplification of high GC content (GC > 60%) DNA sequences. Bovine serum albumin (BSA) has been used as an additive in several applications, including restriction enzyme digestions as well as in PCR amplification of templates from environmental samples that contain potential inhibitors such as phenolic compounds. FINDINGS: Significant increase in PCR amplification yields of GC-rich DNA targets ranging in sizes from 0.4 kb to 7.1 kb were achieved by using BSA as a co-additive along with DMSO and formamide. Notably, enhancing effects of BSA occurs in the initial PCR cycles with BSA additions having no detrimental impact on PCR yield or specificity. When a PCR was set up such that the cycling parameters paused after every ten cycles to allow for supplementation of BSA, combining BSA and organic solvent produced significantly higher yields relative to conditions using the solvent alone. The co-enhancing effects of BSA in presence of organic solvents were also obtained in other PCR applications, including site-directed mutagenesis and overlap extension PCR. CONCLUSIONS: BSA significantly enhances PCR amplification yield when used in combination with organic solvents, DMSO or formamide. BSA enhancing effects were obtained in several PCR applications, with DNA templates of high GC content and spanning a broad size range. When added to the reaction buffer, promoting effects of BSA were seen in the first cycles of the PCR, regardless of the size of the DNA to amplify. The strategy outlined here provides a cost-effective alternative for increasing the efficiency of PCR amplification of GC-rich DNA targets over a broad size range.     

Solid-phase time-resolved fluorescence detection of human immunodeficiency virus polymerase chain reaction amplification products.

A new assay system for the detection of polymerase chain reaction (PCR) amplification products is presented. This single-pot sandwich assay system employs solid-support oligonucleotide-coated capture beads, a rare earth metal chelate-labeled probe, and a time-resolved fluorescence detection. The new assay system was evaluated for various reaction conditions including, DNA denaturation time, hybridization salt concentration, probe concentration, and hybridization time, all of which are important in designing an assay with a high level of sensitivity for the detection of duplex DNA. This nonisotopic assay system was applied to the detection of purified human immunodeficiency virus (HIV) DNA and sensitivity was compared with agarose gel electrophoresis and slot blot hybridization using a 32P-labeled probe. We were able to detect the amplified product from one copy of HIV DNA after 35 cycles of PCR amplification in less than 30 min using this assay, which compared with one copy by gel electrophoresis after 40 cycles of PCR amplification and one copy by slot blot hybridization after 35 cycles of PCR amplification and an overnight exposure of the autoradiogram. Thus, this assay is rapid, sensitive, and easy to use.     

The PCR plateau phase - towards an understanding of its limitations

The DNA polymerases from Thermus aquaticus and Thermus flavus were recently found to bind to short double-stranded DNA fragments without sequence specificity [Kainz et al. (2000) Biotechniques 28, 278-82]. In the present study, it is shown that the accumulation of amplification products during later PCR cycles also exerts an inhibitory effect on several enzymes tested. To simulate later cycle conditions, a 1.7 kb sequence from phage lambda DNA was amplified in the presence of various amounts of a 1 kb double-stranded DNA fragment. A 30-fold molar excess of fragments to polymerase molecules was found to be required for a complete inhibition of Taq, Tfl and Pwo DNA polymerase. This stoichiometric relation remained constant when PCR amplifications were performed using polymerase concentrations of 0.5, 1 or 1.5 U/50 microl reaction volume. The amount of 1 kb DNA fragments required for a complete inhibition was similar to the product yield of the controls (no fragment added), that were run to plateau phase levels. Additionally, PCR mixtures, that were subjected to different numbers of cycles, were compared in their ability to extend 3'-recessed ends by using a hairpin extension assay. The presence of endogenous amplicon DNA accumulated in later PCR cycles was found to inhibit completely the activity of DNA polymerase. PCR mixtures still in quasi-linear phase partially extended the hairpins. In both cases, a further addition of polymerase significantly improved their function. These results indicate that the main factor contributing to the plateau phase in PCR consists of binding of DNA polymerase to its amplification products.     

Detection of Genomic Polymorphism in Plasmodium falciparum Using an Arbitrarily Primed PCR Assay

ABSTRACT. Modifications of the arbitrarily primed polymerase chain reaction assay (i.e. a low annealing temperature and a very slow increase in the temperature during the elongation steps during the amplification cycles) allowed it to be used with the AT-rich Plasmodium falciparum DNA. The analysis of the products by polyacrylamide—urea gels, after silver staining, resulted in high resolution and sensitivity. Eighteen single and six combined pairs of arbitrary primers were tested. Two produced polymorphic patterns complex enough to differentiate between close Colombian isolates in a single assay. This method may be useful in studying the distribution and migration of strains in endemic areas, and for identifying intralaboratory cross-contamination of cultures.     

Extensive Recombination Due to Heteroduplexes Generates Large Amounts of Artificial Gene Fragments during PCR

Artificial recombinants can be generated during PCR when more than two genetically distinct templates coexist in a single PCR reaction. These recombinant amplicons can lead to the false interpretation of genetic diversity and incorrect identification of biological phenotypes that do not exist in vivo. We investigated how recombination between 2 or 35 genetically distinct HIV-1 genomes was affected by different PCR conditions using the parallel allele-specific sequencing (PASS) assay and the next generation sequencing method. In a standard PCR condition, about 40% of amplicons in a PCR reaction were recombinants. The high recombination frequency could be significantly reduced if the number of amplicons in a PCR reaction was below a threshold of 10¹³–10¹⁴ using low thermal cycles, fewer input templates, and longer extension time. Heteroduplexes (each DNA strand from a distinct template) were present at a large proportion in the PCR products when more thermal cycles, more templates, and shorter extension time were used. Importantly, the majority of recombinants were identified in heteroduplexes, indicating that the recombinants were mainly generated through heteroduplexes. Since prematurely terminated extension fragments can form heteroduplexes by annealing to different templates during PCR amplification, recombination has a better chance to occur with samples containing different genomes when the number of amplicons accumulate over the threshold. New technologies are warranted to accurately characterize complex quasispecies gene populations.     

Delineation of type-specific regions on the envelope glycoproteins of human T cell leukemia viruses.

Two different approaches were used to map the type-specific regions on human T cell leukemia virus (HTLV) envelope glycoproteins. 1) Antibody reactivities of polymerase chain reaction-confirmed HTLV-I or HTLV-II carriers' sera were analyzed by Western blot assay with seven recombinant proteins containing different regions of HTLV-I or HTLV-II envelope proteins. 2) Rabbit antibodies elicited by nine HTLV-I Env synthetic peptides were used to react with the native HTLV envelope glycoproteins in an antibody-dependent cellular cytotoxicity (ADCC) assay. The results of the Western blot analysis showed that RP-B2, which contains amino acid residues 166 to 213 from HTLV-II exterior glycoprotein, was specifically reactive with 90.6% (48 of 53) of the HTLV-II carriers' sera but not with any of the HTLV-I carriers' serum (0 of 71). In contrast, RP-B, which contains amino acid residues 166 to 229 from HTLV-I exterior glycoprotein, was reactive with 85.1% (114 of 134) of the HTLV-I carriers' sera but not with any HTLV-II carrier serum (0 of 62). Furthermore, anti-HTLV-I Env synthetic peptide antibody-mediated ADCC identified several distinguishing HTLV-I ADCC epitopes in the middle region (amino acid residues 177 to 257) of the HTLV-I exterior glycoprotein. Therefore, HTLV type-specific epitopes reside mainly in a 69-amino acid sequence bounded by two cysteine residues (amino acids 157 and 225 for HTLV-I and 153 and 221 for HTLV-II), in the middle region of the exterior envelope glycoproteins.