16S rDNA-based Polymerase Chain Reaction Research Articles

Clostridium spp. detection in food samples using 16S rDNA-based PCR method

Raw foods of animal and plant origin are the most likely to be contaminated with either spores or vegetative cells of Clostridium. Molecular detection of the Clostridium spp. by polymerase chain reaction (PCR) is one useful method for detecting Clostridium spp. in some samples such as food raw materials. Clostridium detection was performed using 16S rDNA-based PCR method with Clos 58 - f and Clos 780 - r primers. A study of the specificity of the primers used revealed a reaction to Salmonella and Proteus. Therefore, the scope of PCR with these primers can be food products that have undergone high-temperature processing, for example, sterilized meat or meat and vegetable preserves, as well as ingredients after inactivation of vegetative forms of microorganisms.

Bacterial colonisation of suture material after routine neurosurgical procedures: relevance for wound infection.

Wound healing impairment is a serious problem in surgical disciplines which may be associated with chronic morbidity, increased cost and patient discomfort. Here we aimed to investigate the relevance of bacterial colonisation on suture material using polymerase chain reaction (PCR) to detect and taxonomically classify bacterial DNA in patients with and without wound healing problems after routine neurosurgical procedures. Repeat surgery was performed in 25 patients with wound healing impairment and in 38 patients with well-healed wounds. To determine the presence of bacteria, a 16S rDNA-based PCR detection method was applied. Fragments of 500bp were amplified using universal primers which target hypervariable regions within the bacterial 16S rRNA gene. Amplicons were separated from each other by single-strand conformation polymorphism (SSCP) analysis, and finally classified using Sanger sequencing. PCR/SSCP detected DNA of various bacteria species on suture material in 10/38 patients with well-healed wounds and in 12/25 patients with wound healing impairment including Staphylococcus aureus, Staphylococcus epidermidis, Propionibacterium acnes and Escherichia coli. Microbiological cultures showed bacterial growth in almost all patients with wound healing impairment and positive results in PCR/SSCP (10/12), while this was the case in only one patient with a well-healed wound (1/10). Colonisation of suture material with bacteria occurs in a relevant portion of patients with and without wound healing impairment after routine neurosurgical procedures. Suture material may provide a nidus for bacteria and subsequent biofilm formation. Most likely, however, such colonisation of sutures is not a general primer for subsequent wound infection.

Detection of helicobacter species related genes in mucosa and bile in the primary carcinoma of the gallbladder

Objective To investigate the relationship between helicobacter pylor (Hp) and primary carcinoma of the gallbladder (PCG), providing a theoretical basis for the treatment of PCG.Methods Mucosa and bile of gallbladder samples were collected from 18 patients with PCG (PCG group), 40 patients with chronic cholecystitis or cholelithiasis (CC group), and 20 patients with no PCG and CC (control group). 16S rDNA-based polymerase chain reaction (PCR) followed by DNA sequence analysis of the obtained PCR fragments was performed. A developed search for Hp was also carried out by PCR. Five genes specific for Hp were amplified. Results In the PCG group, 81% samples of mucosa and bile were positive for Helicobacter-specific 16S rRNA gene. The positive rate in PCG group was significantly higher than those of the CC group (50%) and the control group (20%).Of the 16S rDNA sequence of Helicobacter pylori positive samples of mucosa and bile, 52% of samples were positive in cagA, 86% were positive in 26 KDa, 48% were positive in ureA separately. The vacA and rps4 genes were never detected in any of the samples of mucosa and bile. At least one gene was positive in 93% of samples of musca and bile. The sequencing showed more than 99% homology of Helicobacter 16S rRNA in PCG and Hp groups. Conclusion A higher infection rate is present in bile and gallbladder mucosa from patients with PCG. Hp in the biliary system may be one of the risk factors for occurrence of PCG. Key words: Helicobacter pylor; Primary carcinoma of the gallbladder; Polymerase chain reaction

Detection of Bacteroides forsythus and Porphyromonas gingivalis in infected root canals during periapical periodontitis by 16S rDNA

Periapical periodontitis is termed when inflammation of the periodontium is caused by irritants of endodontic origin. Bacterial strains in the root canals were not easy to be identified by the traditional agar culture. In this study a 16S rDNA-based polymerase chain reaction detection method was used to determine the occurrence of Bacteroides forsythus and Porphyromonas gingivalis in chronic periapical periodontitis among Chinese patients. 217 patients with chronic periapcial periodontitis were recruited and a total of 266 teeth were collected. The subjects had no systemic diseases, no antibiotics taken, no root canal treatment (RCT) performed on the infected teeth in the last 3 months. The DNA of bacteria in the root canal was extracted and amplified using universal 16S rDNA primers. The amplification was performed to detect B. forsythus and P. gingivalis using oligonucleotide primers designed from species-specific 16S rDNA signature sequences. B. forsythus and P. gingivalis were detected in 26 and 40% of the participants, respectively. 24 out of 217 infected root canals demonstrated the existence of both types of bacteria, the utility of a 16S rDNA-based PCR detection method showed high sensitivity and high specificity to directly detect B. forsythus, P. gingivalis or other pulpal microorganisms from samples of root canal infections. The results indicated that B. forsythus or P. gingivalis might be a member of the microbiota associated with chronic periapical periodontitis and there was a strong association between the studied species and periodontitis.

Detection of Bacteroides forsythus and Porphyromonas gingivalis in infected root canals during periapical periodontitis by 16S rDNA

Periapical periodontitis is termed when inflammation of the periodontium is caused by irritants of endodontic origin. Bacterial strains in the root canals were not easy to be identified by the traditional agar culture. In this study a 16S rDNA-based polymerase chain reaction detection method was used to determine the occurrence of Bacteroides forsythus and Porphyromonas gingivalis in chronic periapical periodontitis among Chinese patients. 217 patients with chronic periapcial periodontitis were recruited and a total of 266 teeth were collected. The subjects had no systemic diseases, no antibiotics taken, no root canal treatment (RCT) performed on the infected teeth in the last 3 months. The DNA of bacteria in the root canal was extracted and amplified using universal 16S rDNA primers. The amplification was performed to detect B. forsythus and P. gingivalis using oligonucleotide primers designed from species-specific 16S rDNA signature sequences. B. forsythus and P. gingivalis were detected in 26 and 40% of the participants, respectively. 24 out of 217 infected root canals demonstrated the existence of both types of bacteria, the utility of a 16S rDNA-based PCR detection method showed high sensitivity and high specificity to directly detect B. forsythus, P. gingivalis or other pulpal microorganisms from samples of root canal infections. The results indicated that B. forsythus or P. gingivalis might be a member of the microbiota associated with chronic periapical periodontitis and there was a strong association between the studied species and periodontitis.

Improving the specificity of 16S rDNA-based polymerase chain reaction for detecting Borrelia burgdorferi sensu lato-causative agents of human Lyme disease

16S rDNA sequences of Borrelia burgdorferi sensu lato were aligned with the 16S rDNA sequences of Borrelia hermsii, Borrelia turicatae, and Borrelia lonestari in order to identify primers that might be used to more specifically identify agents of human Lyme disease in ticks in human skin samples. Standard polymerase chain reaction (PCR), using an oligonucleotide sequence, designated TEC1, was shown, in combination with a previously developed primer (LD2) to amplify strains of B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii, but not the non-Lyme causing B. hermsii or B. turicatae. This primer pair, designated Bbsl, was successfully used to amplify B. burgdorferi sensu lato from skin biopsies of patients with Lyme disease symptoms as well as from Ixodes scapularis, Amblyomma americanum and Dermacentor variabilis ticks. The primer set Bbsl allows for the rapid detection and differentiation of B. burgdorferi sensu lato from non-Lyme disease-causing Borrelia species in ticks and human tissues. The PCR primer set, Bbsl, will greatly facilitate detection of the causative agents of Lyme disease in infected ticks and human skin samples assisting in epidemiological studies, and potentially allowing for a more rapid diagnosis of the disease in patients.

Detection of identical Helicobacter DNA in the stomach and in the non-cirrhotic liver of patients with hepatocellular carcinoma.

Studies have implied the presence of Helicobacter species in the human liver. The possible relationship between the presence of Helicobacter species in the non-cirrhotic liver of patients with hepatocellular carcinoma (HCC) and the detection of Helicobacter species in their stomach was explored. A 16S rDNA-based polymerase chain reaction (PCR) followed by DNA sequence analysis of the obtained PCR fragments was performed on 51 surgically obtained non-cirrhotic liver specimens and 14 gastric samples. Analysis indicated a significant difference in the presence of Helicobacter-species-specific DNA in the liver of patients with HCC compared with controls. Sequence analysis of these PCR products obtained from HCC patients indicated that they were related most closely to the 16S rDNA sequence of Helicobacter pylori but that they always differed at the same two positions. This same aberrant Helicobacter-species-specific 16S rDNA could be isolated in gastric samples of patients with HCC. These data suggest that gastric colonization with a specific subset of Helicobacter strains is associated with the induction of HCC, either directly via colonization of the liver or indirectly, e.g. via secretion of specific toxins by Helicobacter residing in the stomach.

Peptostreptococcus micros in Primary Endodontic Infections as Detected by 16S rDNA-based Polymerase Chain Reaction

A 16S rDNA-based polymerase chain reaction (PCR) method was used to detect Peptostreptococcus micros in primary root canal infections. Samples were collected from 50 teeth having carious lesions, necrotic pulps, and different forms of periradicular diseases. DNA extracted from the samples was amplified using the PCR assay, which yielded a specific fragment of P. micros 16S rDNA. P. micros was detected in 6 of 22 root canals associated with asymptomatic chronic periradicular lesions (27.3%), 2 of 8 teeth with acute apical periodontitis (25%), and 6 of 20 cases of acute periradicular abscess (30%). In general, P. micros was found in 14 of 50 cases (28%). There was no correlation between the presence of P. micros and the occurrence of symptoms. Findings suggested that P. micros can be involved in the pathogenesis of different forms of periradicular lesions.

Polymerase chain reaction detection of Treponema denticola in endodontic infections within root canals.

A 16S rDNA-based polymerase chain reaction (PCR) method was used to detect the occurrence of Treponema denticola in root canals. Samples were collected from 54 single-rooted teeth having carious lesions and necrotic pulps. DNA extracted from the samples was amplified using the PCR assay, which yielded a specific fragment of T. denticola 16S rDNA. T. denticola was detected in 10 of 29 asymptomatic cases (34.5%), eight of 15 root canals that were tender to percussion (53.3%), and five of 10 teeth with acute periradicular abscess (50%). In general, T. denticola was found in 23 of 54 cases (42.6%). Findings suggest that T. denticola can be involved in the pathogenesis of periradicular lesions of endodontic origin.

Detection of Treponema denticola in endodontic infections by 16S rRNA gene-directed polymerase chain reaction.

A 16S rDNA-based polymerase chain reaction (PCR) method was used to detect the occurrence of Treponema denticola in root canal infections. Samples were collected from 21 single-root teeth having carious lesions, necrotic pulps and radiographic evidences of periradicular bone loss. DNA extracted from the samples was amplified using the PCR assay, which yielded specific fragment of T. denticola 16S rDNA. T. denticola was detected in 11 of 21 cases (52.4%), regardless of the presence or absence of symptoms. Since this spirochete was found in a relatively high percentage of the endodontic infections examined and because it is a pathogenic microorganism involved in periodontal diseases, there are reasons to believe that T. denticola can also participate in the pathogenesis of periradicular lesions of endodontic origin.

Analysis of the 16S rRNA gene sequence of the coryneform bacterium associated with hyperkeratotic dermatitis of athymic nude mice and development of a PCR-based detection assay

By 16S rDNA sequencing the authors have characterized the coryneform bacteria associated with hyperkeratotic dermatitis (HD) of athymic nude mice isolated from six different outbreaks of the disease in Northern Italy. This analysis has allowed the authors to confirm the classification of the bacteria asCorynebacterium bovisand to develop a 16S rDNA-based polymerase chain reaction (PCR) detection assay. The test was performed directly on the DNA extracted from epidermal swabs. The PCR primers were chosen to match the 16S rDNA sequence fragments which differ most from the otherCorynebacteriumspp. The test was shown to be both sensitive and specific forC. bovis. Detection of as few as three viable bacterial cells was possible with the use of an oligonucleotide probe in a liquid hybridization assay.

A 16S rDNA-based PCR method for rapid and specific detection of Clostridium perfringens in food

A 16S rDNA-based polymerase chain reaction (PCR) method was developed for the rapid and specific detection of Clostridium perfringens in food. The PCR primers were designed by a GenBank computer search and they are complementary only with the 16S rRNA gene of C. perfringens by sequence alignment. The PCR product is a 279 BP DNA fragment. All C. perfringens strains tested were positive in the PCR assay and all other species tested were negative, including 11 other species of Clostridium and 38 species of other common bacteria. As few as two cells of C. perfringens in pure culture were detectable. High numbers of other bacterial species did not interfere with the detection of C. perfringens. The PCR amplification required only 30 min to complete. The method can be used for detection of C.perfringens in contaminated food. Samples from 100 g of chicken drumsticks which were inoculated with 20, 200 or 2000 cells of C.perfringens were subjected to the PCR assay and all produced positive results.

Development of PCR-based assays for the detection of two human mollicute species, Mycoplasma penetrans and M. hominis

Recently, a 16S rDNA-based polymerase chain reaction (PCR) assay was developed for the selective and sensitive detection of Mycoplasma pirum. In this study, the same procedure was used in order to selectively detect by PCR two human mycoplasmas, M. hominis and M. penetrans, with a high level of sensitivity even in a context of human DNA. For each assay, the specificity was verified by testing DNA from other mollicute species (including those closely related to the corresponding mycoplasma), from bacteria phylogenetically close to mollicutes, from Escherichia coli and from human peripheral blood mononuclear cells (PBMCs). Each assay proved to be highly sensitive since it reliably detected 10 DNA molecules, even in a context of human DNA. The results of this study demonstrate the suitability of our procedure using primers which were designed for the PCR detection of human mollicutes with a high specificity and a low and reproducible threshold of sensitivity.