One-step Polymerase Chain Reaction Research Articles

FAST, EASY AND EFFICIENT DNA EXTRACTION AND ONE-STEP POLYMERASE CHAIN REACTION FOR THE DETECTION OF XYLELLA FASTIDIOSA IN POTENTIAL INSECT VECTORS

A quick, simple and efficient procedure for detecting Xylella fastidiosa in potential insect vectors is described. The procedure employs a commercially available DNeasy tissue kit for the extraction of high-quality DNA from the insect, followed by one-step polymerase chain reaction amplification using previously published oligonucleotide primers specific to X. fastidiosa. The procedure does not require the use of phenol, chloroform or alcohol for the precipitation of nucleic acids. Also it does not need additional purification or enrichment steps, and can be completed in less than a day. The procedure was used successfully in detecting X. fastidiosa in two potentially important leafhopper species, Graphocephala versuta and G. coccinea, and in a treehopper species Entilia concisa, collected from a nursery where bacterial leaf scorch disease caused by X. fastidiosa occurs.

Expression of cancer-testis antigen (CTA) in tumor tissues and peripheral blood of Chinese patients with hepatocellular carcinoma

To investigate the expression of cancer-testis antigen (CTA) in Chinese patients with hepatocellular carcinoma (HCC), and the relationship between CTA gene expression and clinical indexes, we used one-step reverse transcription polymerase chain reaction (RT-PCR). The expression of the CTA mRNA was investigated in the tissues of HCC and corresponding peripheral blood of 37 patients with HCC. Fifteen samples of cirrhotic tissues and 15 normal tissues were examined with the same method. Two kinds of CTA (SSX-2 and SSX-5) showed high-specific and high-frequent expression in HCC tissues, but neither of them could be detected in adjacent non-HCC tissues. In corresponding peripheral blood of HCC tissues, the positive expression rate of the SSX-2 and SSX-5 mRNA was not very high. No relationship was found between the expression of CTA and clinical indicators such as age, sex, tumor size, TNM staging, serum AFP level and infection with hepatitis virus. In 15 patients with cirrhosis and 15 other non-tumor patients, none of the SSX-2 and SSX-5 mRNA was detected in liver tissue or peripheral blood. High frequency and specificity of CTAs in HCC indicates that their products may be new potential promising targets for antigen-specific immunotherapy of HCC. High frequent co-expression of the two genes in HCC provides a possibility of polyvalent vaccinations for HCC. Specific expression of CTAs was observed in AFP-negative HCC, suggested applying their mRNA as tumor markers to detect circulating HCC cells as adjuvant diagnostic tool and as indicators of recurrence and prognosis.

Rapid detection of Neisseria meningitidis in cerebrospinal fluid by one-step polymerase chain reaction of the nspA gene

A polymerase chain reaction (PCR) protocol for the rapid detection of meningococcal DNA in cerebrospinal fluid (CSF) was developed and optimized. A set of primers based on Neisseria surface protein A ( nspA) gene sequence was designed to amplify a 481-bp product specific for N. meningitidis. We tested 85 N. meningitidis strains obtained from patients with meningococcal meningitis and 112 CSF samples from patients with suspected meningococcal meningitis. No amplification of the nspA gene was observed from other Neisseriaceae species (except from N. gonorrhoeae) and from other bacteria frequently associated with meningitis. N. meningitidis belonging to different serogroups yielded the same product after PCR amplification. The sensitivity and specificity of our protocol was determined by comparing the results of specific amplification of nspA gene by PCR reaction ( nspA-PCR) with those obtained by conventional methods. All positive samples by conventional methods were confirmed by nspA-PCR, whereas 48% of negative samples after culture and latex agglutination tested positive by nspA-PCR. The use of nspA-PCR proved to be a rapid diagnostic method, in which sensitivity and specificity may not be affected by prior antibiotic treatment.

Heparanase-1 gene expression in normal, hyperplastic and neoplastic prostatic tissue

Heparanase-1 (Hpa-1) has been implicated in tumour invasion and metastasis. In the present study, we evaluated the clinicopathological significance of Hpa-1 mRNA expression in prostate cancer and non-cancerous prostatic tissue by one-step polymerase chain reaction (PCR) of laser microdissected prostatic gland cells. In addition, cell type-specific expression of Hpa-1 mRNA in prostatic tissue was analysed by in situ hybridisation. Hpa-1 mRNA expression was found in 50% of normal and 40% of hyperplastic prostatic tissue. In situ hybridisation showed that Hpa-1 mRNA was strongly expressed in prostate gland cells. Of the 26 prostate carcinomas tested, 42% were positive for Hpa-1 mRNA. However, in non-cancerous prostatic tissue, Hpa-1 mRNA was significantly more often expressed than in less differentiated or more invasive prostate cancers (P<0.05). In situ hybridisation revealed only focal Hpa-1 mRNA expression in the neoplastic gland cells. Hpa-1 mRNA expression in the tumours significantly correlated with tumour differentiation and tumour stage (P<0.05). Our data indicate that Hpa-1 gene expression may be lost during dedifferentiation of prostatic gland cells.

Mutational analysis of the human dihydropyrimidine dehydrogenase gene by denaturing high-performance liquid chromatography.

Mutations in the DPYD gene, which encodes dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme in the catabolism of pyrimidines, are responsible for an inborn error of metabolism associated with thymine-uraciluria and neurological symptoms. Because the antimetabolite 5-fluorouracil (5-FU) is metabolized by the same enzyme, deficient DPYD alleles may also constitute a risk factor for severe toxicity following treatment with this anticancer drug. The aim of this study was to develop a comprehensive and rapid method to detect sequence variations within the DPYD gene. Using polymerase chain reaction (PCR) amplification and denaturing high-performance liquid chromatography (DHPLC), we established a protocol that makes it possible to screen all 23 exons of the DPYD gene and their exon-intron boundaries for both known and unknown mutations under identical conditions. A novel one-step PCR mutagenesis procedure was developed to generate heterozygous mutant amplicons as positive controls to optimize DHPLC detection of any sequence variation. DHPLC analysis was shown to result in mutation-specific elution profiles and to be able to distinguish different base changes within the same exon or different heterozygous combinations of mutations within the same exon. By analyzing the DPYD gene in 16 affected individuals, a total of 47 base changes were detected, representing eight known mutations and three novel intronic base changes. Sequence analysis confirmed all base changes detected. This method will be useful in identifying patients at risk for toxicity prior to 5-FU treatment, as well as in the analysis of individual patients with thymine-uraciluria.

Histopathology of Y-organ in Indian white shrimp Fenneropenaeus indicus, experimentally infected with white spot syndrome virus

Histopathology of Y-organ (moulting gland) in Indian white shrimp Fenneropenaeus indicus was studied after challenging the shrimp with virulent white spot syndrome virus (WSSV). Histopathological investigation on the Y-organ collected from moribund shrimps revealed the presence of intranuclear basophilic inclusions, characteristic of WSSV. More than 70% of the Y-organ cells were infected, suggesting the degenerated state of the organ. The cellular integrity of the Y-organ was completely destroyed by the WSSV. Further, Y-organ tissue samples collected from all the 16 experimental shrimp were positive by one-step polymerase chain reaction (PCR), confirming severe WSSV infection. The infected Y-organ in F. indicus with majority of the organ cells observed in the lysed condition suggests a physiological dysfunction of the organ. In uninfected and healthy controls, the lobulated Y-organ showed closely packed normal cells with prominent nuclei and sparse cytoplasm. Physiological implication of a degenerated Y-organ in the moulting and reproduction of the penaeid shrimp is discussed.

Comparison of Different Methods for Preparing Single Stranded DNA for Oligonucleotide Microarray

Single stranded DNAs (ssDNA) were crucial reagents for nucleic acid analysis on oligonucleotide microarrays. Methods for preparing ssDNAs have been reported, but the systematic study about them was still lacking now, especially for oligonucleotide microarrays. Using oligonucleotide microarrays, some of the most popular methods were compared in this report, including asymmetric polymerase chain reaction (PCR), magnetic beads based method, denaturation of double stranded DNA (dsDNA) by heating or by alkaline treatment. All the ssDNA targets prepared by the four methods were applied to oligonucleotide microarrays. The results indicate that the ssDNA targets generated by asymmetric PCR and magnetic beads based method have shown high sensitivity and specificity in hybridization, but those generated by denaturation of dsDNA via heating or alkaline treatment produced serious false negative signals under the same hybridization conditions. Considering the simplicity and the cost of the method, asymmetric PCR is a more favorable approach for oligonucleotide microarrays. In view of the fact that most of the present asymmetric PCR procedures were in two steps, and a purification process was necessary before the second step amplification was carried out, a one-step asymmetric PCR was attempted in this work. The results demonstrate that it was a preferred approach for ssDNA preparation for oligonucleotide microarrays use.

Detection and typing of Helicobacter pylori cagA/ vacA genes by radioactive, one-step polymerase chain reaction in stool samples from children

The detection and molecular typing of Helicobacter pylori virulence genes in human stool specimens by polymerase chain reaction (PCR) require an adequate amount of bacterial DNA and an appropriately adjusted PCR protocol. DNA was isolated from stool samples of 39 H. pylori-infected and nine uninfected Colombian children using the QIAamp Kit following the manufacturer's instructions but with modifications. DNA templates were amplified for the vacA s and m regions and for the cagA gene by PCR using radioactively labeled ( 32P) primers. The modifications in the standard Qiagen protocol of stool DNA extraction increased the final concentration of eluted total stool DNA 4.7 times (117±17 versus 22±3 ng/μl; P<0.0001). Nevertheless, its amplification by regular PCR programs (30–40 cycles) did not generate visible signals because of the very low ratio of H. pylori DNA to other DNA. PCR for 80 cycles successfully amplified vacA in 36/39 samples (sensitivity, 92.3%) and cagA fragments in 21/39 (53.8%) fecal DNA samples. Both s and m vacA regions were amplified in 33/36 (91.7%) DNA samples. The s1m1 genotype was the most commonly isolated variant, accounting for 17/36 or 47.2% of positive samples. The s2m2 genotype was ascertained to be frequent also (14/36 or 38.9%). Almost all (94.1%) s1m1 genotypes were cagA positive. The majority of s2m2 genotypes (78.6%) were not associated with the cagA gene. Neither cagA nor vacA fragments were amplified from DNA isolates of H. pylori-uninfected children nor from DNA isolated from six gastrointestinal bacterial strains (specificity, 100%). The data suggest that the proposed modified technique of DNA extraction and PCR assay of stool samples may be an effective and reliable noninvasive tool for the detection and typing of H. pylori cagA/ vacA virulence genes in infected individuals.

Three-piece-ligation PCR and application in disruption of chlorophyll synthesis genes in Synechocystis sp. PCC 6803.

Linear DNA, consisting of a drug-resistance marker and long flanking sequences, was synthesized by one-step polymerase chain reaction after a three-piece ligating reaction. Chlorophyll synthesis genes, chlH and chlL in Synechocystis sp. PCC 6803, were replaced by a kanamycin-resistance marker through double recombinations with flanking homology regions. Under LAHG conditions, the chlL but not chlH mutant stopped chlorophyll synthesis, while both synthesized chlorophyll in the light.

Nested PCR for diagnosis of tuberculous lymphadenitis and PCR-SSCP for identification of rifampicin resistance in fine-needle aspirates.

An accurate diagnosis of tuberculosis and multidrug resistance is important for the control of tuberculosis, which remains a major public health problem. Fine-needle aspiration (FNA) has provided an alternative tool for bacterial examination. This study was performed to investigate the usefulness of one-step polymerase chain reaction (PCR) and PCR-SSCP as a routine test for the detection of Mycobacterium tuberculosis and rifampicin-resistant strain in FNA. Ziehl-Neelsen stain (Z-N) and PCR were processed using the aspirates of tuberculous lymphadenitis for the detection of M. tuberculosis. PCR-SSCP was done for the identification of rpoB mutation. M. tuberculosis was detected in 49/63 (77.8%) by PCR and 25/63 (39.7%) by Z-N. There were 26 cases with PCR(+)/Z-N(-) and two cases with PCR(-)/Z-N(+). Twelve cases showed negativity against both. In 7/22 (31.8%), rpoB mutation was observed. In conclusion, PCR is more sensitive in the detection of M. tuberculosis in FNA than Z-N. PCR-SSCP could also be used in FNA in the prediction of multidrug resistance.

Rapid detection and identification of Clostridium chauvoei by PCR based on flagellin gene sequence

We developed a one-step polymerase chain reaction (PCR) system that specifically detects Clostridium chauvoei. Oligonucleotide primers were designed to amplify a 516-bp fragment of the structural flagellin gene. The specificity of the PCR was investigated by analyzing 59 strains of clostridia, and seven strain of other genera. A 516-bp fragment could be amplified from all the C. chauvoei strains tested, and no amplification was observed by using DNAs from the other strains tested, including Clostridium septicum. Similarly, this PCR-based method specifically detected C. chauvoei DNA sequences in samples of muscle and exudate of obtained from mice within 12 h of inoculation. In tests using samples of muscle or liver, the limit of detection was about 200 organisms per reaction. These results suggest that the one-step PCR system may be useful for direct detection and identification of C. chauvoei in clinical specimens.

A Highly Specific One-step PCR Assay for the Rapid Discrimination of Enteropathogenic Yersinia enterocolitica from Pathogenic Yersinia pseudotuberculosis and Yersinia pestis

Based on differences within the yopT-coding region of Yersinia. enterocolitica, Y pseudotuberculosis and Y pestis, a rapid and sensitive one-step polymerase chain reaction assay with high specificity for pathogenic Y enterocolitica was developed. By this method pathogenic isolates of Y enterocolitica can be easily identified and discriminated from other members of this genus. The entire coding sequence of the yopT effector gene of Y. pseudotuberculosis Y36 was determined.

Detection of prognostic significant translocations in childhood acute lymphoblastic leukaemia by one-step multiplex reverse transcription polymerase chain reaction.

The search for chromosomal translocations in de novo cases of childhood acute lymphoblastic leukaemia (ALL) is crucial for the selection of the appropriate therapeutic protocol. In this work, we describe a new method - one-step multiplex reverse transcription polymerase chain reaction (RT-PCR) - to screen for prognostic significant translocations in childhood ALL. Our approach involves a single PCR reaction for the simultaneous detection of the molecular rearrangements resulting from the t(9;22), t(12;21), t(4;11) and t(1;19), with a turnaround time of less than 24 h. This assay proved to be highly sensitive, specific, reproducible and easy to implement in a routine genetics laboratory.

Screening of human bladder carcinomas for the presence of Ha-ras codon 12 mutation.

Contradictory results were obtained from previous studies aiming at defining the frequency of Ha-ras codon 12 mutations in bladder tumors. Differences in the sensitivities of the methods used could account for this discrepancy. In this study, we reevaluated the frequency of Ha-ras codon 12 mutations in a series of 87 human bladder tumors using a combination of two different methods. The first was derived from the protocol of Ooi et al and consisted in a one-step allele-specific polymerase chain reaction using mismatched primers in two separate PCR. This method is very rapid and highly sensitive, detecting the presence of minor populations (less than 10%) of mutant alleles. The second strategy consisted in screening all tumors using natural restriction fragment length polymorphism (RFLP) analysis. The two methods were in complete concordance and enabled us to show that only one out of 87 primary bladder carcinomas (1%) exhibited the mutation, in accordance with previous studies. These results strongly suggest that, even if minor cell populations overexpress codon 12 Ha-ras mutation, the analysis of this mutation cannot be used to screen potentially invasive transitional cell tumors of the bladder.

Cost-effective screening methods for various single gene defects in single cells using high magnesium and total ionic strength and restriction enzymes.

A reliable cost-effective protocol for the diagnosis of various defective genes in single blastomeres from preimplantation embryos has been established. Single cells were lysed in alkali buffer followed by neutralization and addition of a solution containing a high concentration of sulfhydryl reducing agents and MgCl(2) in relatively high ionic strength (0.45) (solution M) with or without restriction enzyme(s). The reaction mixture was incubated at 37 degrees C for 15 min followed by heat denaturation at 95 degrees C for 10 min. Respective polymerase chain reaction (PCR) mixture was then added to amplify each designated DNA region. The treatment of neutralized single cell lysate with adequate restriction enzyme(s) which do not cleave the target DNA sequences but shortens the genomic template DNA strands. This may facilitate primer-template annealing. The subsequent heat denaturation of the cell lysate in solution M indeed gave better signals of amplified DNA fragments on polyacrylamide gels. Defects in Tay Sachs exons 11 and 12, CF-DeltaF508 and CF-N1303K, and genomic sequences of ZFX/ZFY were successfully detected on gels after one-step PCR amplification, especially those cell lysates treated with restriction enzymes. In conclusion, a cost-effective one-step PCR method for amplifying various specific genomic regions containing a single gene defect in single cells has been established. This protocol may be applied to genetic screening for many single defective genes of biopsied single blastomeres from preimplantation in vitro fertilization (IVF) embryos.

One-step heminested PCR for amplification ofNeisseria meningitidis DNA in cerebrospinal fluid

A one-step polymerase chain reaction (Heminested-PCR) was designed to target the 16S rRNA fragment simultaneously using a set of primers for the universal bacterial group and a Neisseria meningitidis species-specific sequence for diagnostic purposes. The diagnostic features of the Heminested-PCR were evaluated in the study of 168 cerebrospinal fluid (CSF) specimens from 84 patients with a N. meningitidis infection, meningitis caused by unrelated bacteria and other etiologies (57 patients), or suspicious cases (27 patients) with clinical symptoms of bacterial meningitis but with negative results from bacteriological procedures. About 90% of patients with bacterial meningitis, including those suspicious cases, had prior antibiotic therapy. The sensitivity, specificity, positive, and negative predictive values found in relation to culture and/or microscopy were 91.7, 100, 100, 100, and 90.5%, respectively. In patients suspected of having bacterial meningitis, the Heminested-PCR revealed 51.9% (14 patients) positive for N. meningitidis infection and 40.7% (11 patients) positive for unrelated bacterial infections. The agreement of the Heminested-PCR with culture and/or microscopy was high and ranked as almost perfect (kappa indices > 0.856), in contrast to its agreement with other techniques. These findings speak in favor of the molecular diagnosis of meningococcal meningitis in patients who are culture- and/or microscopy-negative, due to their prior antibiotic treatment.

Relative Quantitation of mRNA in β-Thalassemiamb E Using Real-Time Polymerase Chain Reaction

β-Thalassemia and Hb E patients, with seemingly identical genotypes, have a remarkable variability in severity. Reduction in red cell survival in β-thalas-semia is correlated with the amount of intracellular unmatched α-globin chains. However, it was only recently realized that mRNA, whose translation is prematurely terminated, is also unstable. No systematic attempts have been made to investigate mRNA stability in β-thalassemia arising from nonsense mutations located upstream from the normal termination codon. In this study, one-step real-time polymerase chain reaction has been employed to compare the levels of α- and β-globin mRNA in reticulocytes from β-thalassemia/Hb E subjects. The results showed the highest α/β-globin mRNA ratio (median = 5.70, n = 13) in frameshift codons 41/42 (-TTCT)/Hb E individuals compared to normal subjects (median = 1.02, n = 6), or those with Hb E trait (median = 2.15, n = 8). In addition, there was a concomitant increase in the α/β-globin mRNA ratio with decrease in hemoglobin level, ie., increase in severity. The difference in the ratio among β-thalassemia/Hb E patients with the same genotype may be attributed to individual variations of efficiency in βE-globin mRNA splicing and in the destruction of prematurely terminated mRNA.

One-step polymerase chain reaction-based typing of Helicobacter pylori vacA gene: association with gastric histopathology.

Heterogeneity of the Helicobacter pylori vacA gene may be associated with bacterial virulence and presentation. In this study, the possible correlation between vacA genotypes and gastric histopathology was investigated. Using a modified one-step polymerase chain reaction (PCR)-based method, 122 of 131 H. pylori isolates obtained from 63 of 67 patients from Germany were classified into distinct vacA genotypes according to their signal sequence (s1 or s2) and their midregion alleles (m1 or m2). A possible subtype of ml, now alluded to as m3, was identified in one-third of the isolates. Signal sequence s1 was significantly associated with higher H. pylori density but not with gastric inflammation parameters as compared with s2. Compared with m2, ml initially appeared to correlate with higher mononuclear cell scores in corpus, although not with H. pylori density. Upon differentiation between ml and m3, however, only the latter was associated with the high cell scores. Moreover, m3 also correlated with a higher antral H. pylori density. Positive cagA status correlated significantly with vacA signal sequence s1, and higher gastric mononuclear cell scores and corpus neutrophil score. H. pylori density was always associated with enhanced gastric neutrophil and corpus mononuclear cell scores. These data indicate a significant association of specific vacA genotypes with enhanced bacterial density and gastric inflammation. PCR-based identification of the respective alleles can now easily be performed in the diagnostic laboratory using a one-step PCR assay.

A polymerase chain reaction protocol for the detection of various geographical isolates of white spot virus

Polymerase chain reaction (PCR) primers were designed based on the sequence of a cloned fragment of the white spot virus (WSV) genome and were used to detect at least four geographic isolates of WSV from both experimentally-and naturally-infected shrimp. In addition to high specificity, the one-step and two-step PCR protocols were determined to have sensitivities of 10–100 pg and 100 femtograms respectively. The two-step PCR protocol is recommended as a very sensitive and specific alternative protocol to Western blot assay for the detection of WSV.

Low frequency of CYP2A6 gene polymorphism as revealed by a one-step polymerase chain reaction method.

Human cytochrome P450 2A6 (CYP2A6) has been shown to metabolically activate carcinogens and mutagens. Genetic polymorphisms for CYP2A6 have been reported previously in different ethnic groups using a two-step polymerase chain reaction (PCR) method to identify CYP2A6*1, CYP2A6*2 and CYP2A6*3. Moreover, a new truncated allele has been recently identified in a Japanese population. We report here a one-step PCR amplification of the CYP2A6 gene from human genomic DNA and the detection of intact CYP2A6 alleles by restriction enzyme digestion. The diagnostic exon (exon 3) of the CYP2A6 gene was amplified from human genomic DNA with a primer pair. The forward primer is unique to the CYP2A6 gene, which eliminates previous problems in amplifying two highly homologous CYP2A genes, CYP2A7 and CYP2A13, in humans. The resulting PCR products (214 bp) were digested with XcmI or DdeI to detect the presence of CYP2A6*2 or CYP2A6*3 alleles, respectively. The allelic frequencies for CYP2A6*2 were 2.3% (n = 320) in the Caucasian and 0.7% (n = 71) in the Chinese populations, respectively. CYP2A6*3 allelic frequency in the Chinese population was 0.7%; while no CYP2A6*3 allele was detected in the Caucasian population. The allelic frequencies are relatively low and the reason for this discrepancy between different methods is discussed.