Evaluation of five DNA extraction methods for detection of H. pylori in formalin-fixed paraffin-embedded (FFPE) liver tissue from patients with hepatocellular carcinoma

Since Helicobacter spp. DNA was identified in liver tissue resected from patients with hepatocelullar carcinoma (HCC), researchers have suggested a role of this bacterium in hepatic carcinogenesis. Archives of formalin-fixed, paraffin-embedded (FFPE) tissues represent an extraordinary source for clinical studies providing many advantages. However, DNA extraction from FFPE tissues is laborious, time-consuming and still remains a challenge. The aim of this study was to evaluate five protocols for DNA extraction from FFPE liver obtained from patients with HCC in order to detect Helicobacter pylori DNA. These methods were: (1) QIAamp FFPE Tissue Kit, (2) QIAamp DNA Mini Kit, (3) Wizard SV Genomic DNA Purification System, (4) RealiaPrep FFPE gDNA Miniprep System and (5) phenol–chloroform. H. pylori detection was performed using 16S rRNA gene amplification by PCR. The highest total amount of DNA was obtained using the phenol–chloroform method. Analyses of 16S rRNA gene amplification did not show statistically significant differences among the methods (p = 0.466), although the highest percentage of positive cases (70%) was found in samples extracted with phenol-chloroform. We suggest that of the five methods evaluated, phenol/chloroform is the most suitable for detection of H. pylori in FFPE liver from patients with HCC.     

Colorimetric one-tube nested PCR for detection of Trichomonas vaginalis in vaginal discharge.

A colorimetric one-tube nested PCR was developed for the detection of Trichomonas vaginalis in clinical vaginal discharge specimens. A family of 650-bp specific DNA repeats from the T. vaginalis genome was targeted. There was no cross-reaction with human DNA or other infectious agents, including Pentatrichomonas hominis and Giardia lamblia. The colorimetric assay was applied as an adjunct to nested PCR for semiquantitative determination of T. vaginalis DNA at levels corresponding to 1 to 1,000 parasites. PCR of samples prepared by a rapid boiling method was as sensitive and specific as PCR of samples prepared by the standard DNA extraction method: the equivalent of one T. vaginalis organism in 20 microliters of vaginal discharge could be detected. The colorimetric nested PCR was compared with wet mount and culture for the detection of T. vaginalis. A total of 378 clinical vaginal discharge specimens from symptomatic patients were examined; 31 patients were positive for T. vaginalis both by culture and by nested PCR. However, only 17 of these 31 patients were positive by wet mount examination. In addition, of 113 asymptomatic patients, 9 were positive for T. vaginalis by nested PCR. Of these nine PCR-positive patients, only two were also positive both by wet mount and by culture, four patients were positive by culture but negative by wet mount, and three patients were negative both by wet mount and by culture. No specimens negative by nested PCR were positive by wet mount or by culture. The three asymptomatic patients with PCR-positive but wet mount- and culture-negative samples were subsequently found to have T. vaginalis infection after repeated and prolonged culture was performed. This colorimetric nested PCR was very sensitive compared with culture for the diagnosis of vaginal trichomoniasis, especially asymptomatic T. vaginalis infection. It is also simple, specific, rapid, and semiquantitative.     

Helicobacter pylori in a costa rican dyspeptic patient population

Gastric biopsies from 65 Costa Rican dyspeptic patients were investigated for the presence ofHelicobacter pylori DNA by polymerase chain reaction (PCR) amplification of 16S rRNA sequences. Both frozen and paraffin-embedded samples were used, and the results were compared with bacterial culture and histological examination.Helicobacter pylori DNA was detected by PCR in 60 (92 %) of the patients, andHelicobacter pylori strains confirmed by PCR could be isolated from 37 of them. Altogether, 59 patients were shown to be infected using the combination of culture and histology as the reference method. The sensitivity of PCR analysis of frozen material was 98 % (58/59). The PCR analysis of paraffin-embedded samples seemed less reliable than that of frozen biopsy material.     

Evaluation of extraction methods from paraffin wax embedded tissues for PCR amplification of human and viral DNA

AIM: To evaluate the efficiency of phenol/chloroform, microwave, and Qiagen spin column based DNA extractions from paraffin wax embedded tissue for use in the polymerase chain reaction (PCR). In addition, to assess the reliability of amplifying a housekeeping gene to indicate successful viral DNA extraction. METHODS: DNA samples extracted from 20 blocks of cervical carcinoma tissues using the three methods were subjected to PCRs targeting 509 bp and 355 bp of the beta globin gene, and 450 bp and 150 bp of human papillomavirus (HPV) DNA. RESULTS: Microwave extraction showed the highest positive rate for beta globin PCR, whereas the spin column method was the most efficient for HPV DNA extraction. When the 509 bp beta globin and 450 bp HPV PCR results were correlated, two of 10, eight of 12, and nine of 10 beta globin positive extractions prepared by means of the phenol/chloroform, microwave, and spin column methods, respectively, yielded HPV DNA of the expected size. For the beta globin negative samples, HPV was detected in three of 10, two of eight, and four of 10 samples. CONCLUSIONS: HPV DNA extraction was most efficient using the Qiagen spin column and had the highest positive predictive value when a housekeeping gene was used as an indicator of successful viral DNA extraction; the phenol/chloroform method was the least efficient. The potential drawbacks of some extraction methods when using a human housekeeping gene to assess the quality of viral DNA extraction need to be considered.     

Presence ofHelicobacter pylori in the oral cavity,oesophagus, stomach and faeces of patients with gastritis

The presence ofHelicobacter pylori in the oral cavity (6 sites), oesophagus, stomach and bowel of 20 dyspeptic patients was investigated. Samples were cultured on three selective media and analyzed by 16S rDNA polymerase chain reaction (PCR) and southern hybridization.Helicobacter pylori DNA was detected by PCR from oral-cavity samples of three (20 %) and from faeces samples of only one (7 %) of the patients whose stomach biopsies were positive forHelicobacter pylori. When culture was used, the microorganism's rate of recovery from the oral cavity and faeces was 13 % and 7%, respectively. One patient had aHelicobacter pylori-like organism in samples collected from the tongue and palate. Both strains were urease, catalase and oxidase positive and grew microaerophilically but were negative on PCR analysis. This demonstrates the possibility of false identification ofHelicobacter pylori by use of routine enzyme reactions. Interestingly, specimens collected from the cheeks of three patients were positive forHelicobacter pylori by PCR analysis. This is the first instance of detection of this micro-organism in the cheek.     

Development of a rapid PCR method for identification ofHelicobacter pylori in dental plaque and gastric biopsy specimens

A rapid and simple polymerase chain reaction (PCR) method was developed to detectHelicobacter pylori in gastric biopsy specimens and dental plaque samples. The primers were targeted to the 16S rRNA sequence ofHelicobacter pylori strain ATCC 43504. The system was found to have a theoretical detection level of 0.5 to 5Helicobacter pylori cells in a 5 l sample of dental plaque. In the absence of plaque, the detection level was even better: theoretically, 0.05 to 0.5Helicobacter pylori cells were detected in water suspension. However, this appeared to be due to the presence of free bacterial DNA in the culture used for the sensitivity determination. Thus, the actual sensitivity of the system was found to be fewer than fiveHelicobacter pylori cells, irrespective of the type of sample used. The method was then used to analyse 29 dental plaque and gastric biopsy specimens collected from patients with a history of recurrent peptic ulcer disease. Fourteen stomach specimens were positive forHelicobacter pylori when tested with the PCR method, while the respective figures with culture, histological examination and the urease test were 11, 12 and 9. No positive dental plaque samples were observed.     

Use of magnetic beads for tissue DNA extraction and IS6110 Mycobacterium tuberculosis PCR.

Polymerase chain reaction (PCR) techniques are used increasingly for the diagnosis of Mycobacterium tuberculosis infection and can be used on the DNA obtained from both frozen and formalin fixed, paraffin wax embedded tissues. However, the extraction of DNA by means of the conventional phenol/chloroform method is time consuming and requires the use of potentially dangerous chemical reagents. This paper describes a method based upon the use of magnetic beads for the extraction of M tuberculosis DNA from both routinely formalin fixed, paraffin wax embedded tissues and frozen tissues. Magnetic bead extracted DNA from brain, lymph node, and lung tissues collected from patients with human immunodeficiency virus and tuberculosis was compared with that extracted using the phenol/chloroform method. The magnetic bead extraction procedure requires less than two hours, including the time necessary to dewax the tissue sections. In all cases, the DNA extracted with both methods was amplified successfully by PCR for the M tuberculosis IS6110 sequence. Magnetic bead DNA extraction can be used on both frozen and archival tissues: the method is reliable, simple, sensitive, and rapid; in addition, it does not use hazardous procedures or specialised laboratory equipment and can be used for routine DNA isolation from various human tissues.     

Use of magnetic beads for tissue DNA extraction and IS6110 Mycobacterium tuberculosis PCR.

Polymerase chain reaction (PCR) techniques are used increasingly for the diagnosis of Mycobacterium tuberculosis infection and can be used on the DNA obtained from both frozen and formalin fixed, paraffin wax embedded tissues. However, the extraction of DNA by means of the conventional phenol/chloroform method is time consuming and requires the use of potentially dangerous chemical reagents. This paper describes a method based upon the use of magnetic beads for the extraction of M tuberculosis DNA from both routinely formalin fixed, paraffin wax embedded tissues and frozen tissues. Magnetic bead extracted DNA from brain, lymph node, and lung tissues collected from patients with human immunodeficiency virus and tuberculosis was compared with that extracted using the phenol/chloroform method. The magnetic bead extraction procedure requires less than two hours, including the time necessary to dewax the tissue sections. In all cases, the DNA extracted with both methods was amplified successfully by PCR for the M tuberculosis IS6110 sequence. Magnetic bead DNA extraction can be used on both frozen and archival tissues: the method is reliable, simple, sensitive, and rapid; in addition, it does not use hazardous procedures or specialised laboratory equipment and can be used for routine DNA isolation from various human tissues.     

Comparison of polymerase chain reaction with standard methods in the diagnosis ofMycobacterium tuberculosis infection

The polymerase chain reaction (PCR) method was evaluated in the detection ofMycobacterium tuberculosis in comparison with direct microscopy and culture procedures including the standard radiometric system (Bactec). Amplified DNA fragments from clinical samples were analyzed by dot-blot and non-radioactive oligonucleotide hybridization techniques or by agarose gel electrophoresis. The results revealed nested PCR to be the method of choice. The combination of three culture methods could detectMycobacterium tuberculosis in only 79 % of PCR-positive cases. The mean time required to achieve a positive culture result was 17 days in contrast to the PCR method requiring only two to three days. The nested PCR assay provided rapid and reliable results allowing a definitive diagnosis particularly in a number of samples which were negative on culture.     

Quantitative polymerase chain reaction for the detection ofHelicobacter pylori in gastric biopsy specimens

A variety of methods, including the polymerase chain reaction (PCR), are available for the detection ofHelicobacter pylori in clinical samples, but none of them can adequately quantify the organism. In the present study, the competitive PCR, a rapid and simple method for quantification ofHelicobacter pylori DNA in gastric biopsies, was used to measure the amount of DNA present inHelicobacter pylori-positive biopsies. This method is based on coamplification of an internal standard and a target DNA sequence with one set of primers. The internal standard was prepared using a nonhomologous fragment of DNA ligated to specific primers used to amplify the target DNA. This competitive DNA fragment of a desired size and containing primer templates is called a PCR MIMIC. To perform a quantitative PCR, PCR amplification reactions were spiked with known quantities of PCR MIMICs containing unknown amounts of DNA fromHelicobacter pylori-positive biopsies. The amount of target DNA was determined by visual comparison of the PCR products after establishment of the correlation between the internal control concentration and the DNA concentration in a competitive amplification reaction. The results were confirmed by a radioactive method. Quantitative PCR can be a reliable method for determining the extent ofHelicobacter pylori infection.     

Evaluation of a Helicobacter pylori Stool Antigen Test for the Diagnosis and Follow-Up of Infections in Children

 The aim of this study was to evaluate the performance of a newly developed enzyme immunoassay kit (HpSA) for detecting Helicobacter pylori antigens in the stool of children. This study was comprised of 58 children referred to various endoscopy units for evaluation of gastrointestinal symptoms and upper gastroduodenal endoscopy and 11 children for post-therapy follow-up. In the first group, 23 children were diagnosed as positive for Helicobacter pylori using bacteriological and/or histological methods. Stool antigens were detected in 20 of these positive patients, for a sensitivity of 86.9% and a negative predictive value of 91.9%. Since only one false-positive reaction was observed with the HpSA kit, the specificity was 97.1% and the positive predictive value 95.2%. Results obtained for post-therapy follow-up were also promising. The HpSA assays were negative for the eight children whose infections were eradicated after therapy, and a positive result was obtained for two of three patients who had a persistent infection.     

A nested polymerase chain reaction for detection of Legionella pneumophila in clinical specimens

Objective: Because presently used methods for diagnosis of Legionella pneumonia lack sufficient sensitivity and sometimes specificity and rapidity, the detection of Legionella spp. by amplification of nucleic acids might be valuable. However, performing polymerase chain reaction (PCR) on clinical samples such as sputum is difficult because of the presence of extraneous DNA and inhibitors of the reaction. An attempt to circumvent these problems was made.
Method: A nested PCR method was devised using primers from the mip gene of Legionella pneumophila. This PCR was tested on pure cultures of legionellae and clinical isolates of other bacteria. Clinical samples (bronchoalveolar lavage fluid, bronchial aspirate and sputum) from patients who suffered from legionellosis and samples from patients who suffered from other causes of pneumonia were also tested.
Results: The PCR was specific for L. pneumophila and no non- Legionella bacteria reacted. Ten to 50 colony forming units of Legionella in the sample could be detected. Twenty-two of 25 clinical samples were positive among patients suffering from pneumonia proven to be due to L. pneumophila serogroups 1, 3, 4, 5 and 6. Two of the three negative samples were from patients who had been treated with adequate therapy for at least 2 days and were culture negative. However, nine other culture-negative samples were PCR positive, of which seven came from patients who had been treated for 3–7 days. All pneumonia patients in the control group proved negative in PCR. A commercial kit for DNA preparation from clinical samples, based on absorption of nucleic acids to silica gel, was superior to the traditional phenol/chloroform extraction and increased the rapidity, simplicity and sensitivity of the procedure.
Conclusions: A nested, simplified and rapid PCR method using mip primers proved to be more sensitive than culture and as sensitive and specific as other PCR procedures previously reported.     

Alteration in sample preparation to increase the yield of multiplex polymerase chain reaction assay for diagnosis of genital ulcer disease

Purpose: Genital Ulcer Disease (GUD) is common sexually transmitted infection (STI). Multiple studies have shown that GUDs are strongly associated with the transmission and the acquisition of HIV infection. An accurate diagnosis of common etiology of GUD namely Herpes, syphilis and Chancroid is possible using Multiplex PCR (M-PCR). However, frequent presence of Polymerase Chain Reaction inhibitors in the ulcer swab specimen limits the performance of the assay. In order to overcome this problem, alternative specimen preparation method was used. Materials and Methods: To determine the common etiology, GUD specimens obtained under an STI operations research study were tested with M-PCR after the samples were prepared using Roche Amplicor specimen preparation kit. PCR inhibiting samples were identified from that, which showed negative results. These samples were subjected to phenol-chloroform extraction and ethanol precipitation before the conduct of M-PCR on them. Results: Of the 237 GUD specimens tested, in 145 etiologies could be detected, whereas 92 samples were found negative. Further spiking with one of the target DNA, 128 of the negative samples were found to contain the inhibitors. These 126 samples were then subjected to phenol chloroform extraction and ethanol precipitation followed by M-PCR. Using this method for sample preparation, etiology could be determined in 46 (23%) additional samples. This success rate of altered sample preparation method has been lower than that has reported. Conclusion: The results indicate that sample preparation using phenol chloroform extraction and ethanol precipitation, prior to M-PCR helps to eliminate the inhibitors and increase the yield of the assay. However, being a laborious procedure, it may be used for samples giving negative results after the screening by Roche Amplicor specimen preparation kit.     

Use of a simple DNA extraction method for high-throughput detection of filarial parasite Wuchereria bancrofti in the vector mosquitoes

Molecular xenomonitoring of filariasis is the detection of filarial DNA in mosquitoes by PCR and a useful tool for monitoring transmission. DNA extraction coupled with PCR allows rapid detection of the presence or absence of the filarial parasite in vector mosquitoes compared to traditional method of manual dissection of the mosquito and observation for parasite under a microscope. A Tris–EDTA (TE) buffer-based boiling method of DNA extraction developed earlier by us was employed and explored for its suitability in the detection of Wuchereria bancrofti DNA in pools of Culex quinquefasciatus mosquitoes in real-time PCR assay. In this preliminary study, 1,000 laboratory-reared C. quinquefasciatus were made into 40 pools, each containing 25 mosquitoes spiked with 2mf. DNA from the first 20 pools was extracted using Qiagen DNeasy blood and tissue kit as standard, and the other 20 pools were subjected to TE buffer-based boiling method of DNA extraction. When the results (Ct values) obtained for DNA samples extracted by TE buffer-based boiling method were compared with that of the DNA samples extracted by the standard Qiagen method, they were found to be highly concordant without any significant difference (P?=?0.9). Besides being cost- and time-effective, this protocol was found useful in extracting filarial DNA from two other mosquito genus Aedes and Anopheles, species of which have been reported as important vectors of W. bancrofti in other endemic regions of the world. Thus, TE buffer-based boiling method of DNA extraction is useful for the high-throughput detection of W. bancrofti in vector mosquitoes.     

Effect of specimen collection techniques,transport media,and incubation of cultures on the detection rate ofHelicobacter pylori

Culture and histologic examination are considered gold standard methods for the detection ofHelicobacter pylori, but discrepancies may occur with either method. Failure to detectHelicobacter pylori may be due to sampling error, inappropriate transport or culture media, or insufficient duration of the incubation period. Rates of detection ofHelicobacter pylori by culture and histopathologic examination of gastric mucosal biopsy specimens were determined in 102 consecutive dyspeptic patients. In a separate group of 60 patients, rates of detection ofHelicobacter pylori by culture of antral brushings and the length of incubation required in selective and nonselective culture media were studied. In the first group of 102 patients, the combination of culture and histologic examination detected 54Helicobacter pylori-positive patients, whereas the separate techniques each detected 51Helicobacter pylori-positive patients. In the second group of 60 patients evaluated by culture of antral brushings, the rate of detection ofHelicobacter pylori was 25 of 60 and was similar for culture (25/60) and histologic examination (25/60). In the second group the length of incubation required to detectHelicobacter pylori was different for selective and nonselective media. In nonselective media, incubation of up to ten days was required to detect allHelicobacter pylori infections, whereas in selective media seven days was sufficient. Rates of detection ofHelicobacter pylori by culture, histopathologic examination and culture from brushings were similar, whereas the combination of culture and histopathologic examination achieved a superior rate of detection. The incubation period required for the detection ofHelicobacter pylori by culture was a minimum of seven days and was dependent on the culture medium used.     

两种全血基因组DNA提取法的比较

目的比较经典的酚／氯仿法和人全血基因组DNA小量快速提取试剂盒抽提法两种方法提取人全血基因组DNA的纯度及总量的差异。方法收集静脉血5毫升,共132人份。分别采用上述两种方法提取基因组DNA,用紫外分光光度仪及琼脂糖凝胶电泳检测其纯度和总量,采用统计学t检验,数据以均数±标准差（x±s）表示。结果经典的酚／氯仿法和人全血基因组DNA小量快速提取试剂盒抽提法两种方法提取基因组DNA纯度用0D260／OD280表示分别为：1．65＋0．12,1．86＋0．15。基因组DNA总量分别为28．3＋3．02,33．8＋3．24。结论从提取的基因组DNA纯度和总量来比较,人全血基因组DNA小量快速提取试剂盒抽提法抽提方法较好。     

Comparison of hepatitis B virus subtyping of d/y determinants by radioimmunoprecipitation assay and the polymerase chain reaction.

Using a double polymerase chain reaction a method was devised for detecting and subtyping hepatitis B virus DNA in serum samples. Primers from the S-gene were selected from the sequence analyses of five HBV HBsAg subtypes, to amplify HBV DNA and subtype for y specific DNA. Thirty-eight samples were subtyped for d and y determinants by radioimmunoprecipitation assay (RIPA) and the polymerase chain reaction (PCR). Subtyping by PCR and RIPA was in agreement in 100% of subtype y samples and 83.3% of subtype d, giving an overall correlation of 92.1%. As a third comparison, 12 amplified samples were digested by the restriction enzyme Sau 3A, which differentiates between subtypes y and d. The digest results agreed with PCR in 83.3% of the samples. In addition, we compared our standard phenol/chloroform extraction against a rapid one step method. The phenol/chloroform stage was found to be essential for the removal of nucleases and polymerase inhibitors present in sera.     

Altered Th17 Cytokine Expression in Helicobacter pylori Patients with TLR4 (D299G) Polymorphism

Helicobacter pylori (H. pylori) is associated with gastric ulcer and gastric adenocarcinoma. Polymorphisms in the host genes coding for Toll-like receptors (TLRs) may influence the innate and adaptive immune response to the infection, affecting the susceptibility to H. pylori or the disease outcomes. However, the details and association with different polymorphism and clinical expression of infection remain unclear. A case-control study consisting of 58 patients with H. pylori infection and 44 H. pylori uninfection was conducted. Genomic DNA was extracted and genotypes of TLR4 Asp299Gly polymorphism were assessed through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Mucosal cytokines expression in H. pylori-infected and uninfected gastric biopsies was determined by real-time PCR. The expression of IL-6, IL-17, IL-21, IL-23 and TGF-β1 was signi?cantly higher in patients with D299G polymorphism in TLR4. But the expression of IL-18 between patients with single-nucleotide polymorphisms (SNPs) in TLR4 and patients with the wild-type allele was not signi?cant. In H. pylori-infected patients with gastritis, SNPs in TLR4 may alter cytokine expression toward Th17 immune response in the gastric mucosa and may have increased risk for the development of peptic ulcer.     

Detection of Aspergillus DNA in cerebrospinal fluid from patients with cerebral aspergillosis by a nested PCR assay

Invasive aspergillosis (IA), a complication with high mortality rates, especially in disseminated IA with cerebral involvement, is difficult to diagnose. Biopsy of cerebral lesions is often not feasible, and culture of Aspergillus spp. from cerebrospinal fluid (CSF) is frequently negative. New molecular methods have emerged for diagnosing IA. So far, there are only few reports of Aspergillus DNA detection in CSF. After modifying the DNA extraction protocol, we detected Aspergillus DNA in CSF samples by a previously described nested PCR assay. In six patients with hematologic malignancy and cerebral aspergillosis, CSF samples were investigated for Aspergillus DNA. IA was classified according to the EORTC/MSG 2002 criteria. Two patients each had proven, probable, and possible IA. Thirty-five CSF samples were investigated for Aspergillus DNA by nested PCR. Samples with positive results in the nested PCR assay were quantified by LightCycler PCR assay. Fourteen CSF samples showed positive results in the nested PCR assay. Of these, six samples gave positive results in real-time PCR. The range of CFU per ml was 2,154 to 63,100,000. The highest number of CFU per ml was found in a CSF sample of a patient with acute lymphocytic leukemia and probable cerebral aspergillosis. Detection of Aspergillus DNA in CSF samples is thus possible and has the potential to improve diagnosis of cerebral aspergillosis. Further prospective studies with larger numbers of patients must be performed to evaluate the clinical significance of Aspergillus PCR with CSF samples.     

Antigenicity of fractions ofHelicobacter pylori prepared by fast protein liquid chromatography and urease captured by monoclonal antibodies

The antigenicity ofHelicobacter pylori protein fractions separated by fast protein liquid chromatography size exclusion was investigated by EIA with sera from patients of well definedHelicobacter pylori status. The antigenic material ofHelicobacter pylori was confined to fractions 8 and 14 to 21. Urease containing fractions (14/15) and flagella containing fractions (17/18) were identified. Fraction 8 non-specifically bound human immunoglobulin as demonstrated by the binding ofHelicobacter pylori negative sera. The remaining fractions 14 to 21 when used individually as EIA antigens were 91–100 % specific, however fractions 16 to 19 showed a reduced sensitivity (78 %) compared with the acid extract (95 %). The urease fractions were 91 % sensitive. Purified urease antigen captured by antiurease monoclonal antibodies was 83 % sensitive and 93.3 % specific.