Comparison of intestinal microbial community succession based on different universal primer sets / 生物工程学报

The important role of intestinal microorganisms in human health has been widely confirmed. At present, most of the studies on intestinal microorganisms are based on amplification of the V3-V4 region of bacterial 16S rRNA gene, and little attention has been paid to archaea. In this study, a primer set which can amplify 16S rRNA gene of both bacteria and archaea at the same time was used. By comparing the community changes before and after probiotics intake, it showed that this primer set is suitable for analyzing the changes of human intestinal bacteria and archaea communities. The fecal samples of volunteers were collected, and the amplification and high-throughput sequencing were carried out by using bacterial primer set (B primer) and bacterial and archaeal universal primer (AB primer); several commonly used rRNA databases were used to determine the amplification ability of the primer set to bacteria and archaea. The results showed that AB primer could display the bacterial community amplified by B primer, and could obtain the sequence of common methanogenic archaea in intestinal tract. AB primer set can analyze the bacteria and archaea in the intestinal tract at the same time by only one amplification and sequencing, which can show the structure of intestinal microbial community more comprehensively, which is suitable for the research of intestinal microorganisms.

Value of universal primer PCR for diagnosing bacterial and fungal infec-tion of central nervous system / 中国感染控制杂志

Objective To understand pathogen spectrum of bacterial and fungal infection of central nervous system (CNS),and evaluate the etiological diagnostic value of universal primer polymerase chain reaction (PCR).Methods Data about patients with suspected or confirmed bacterial and fungal infection of CNS from January 2009 to March 2015 were collected,species of pathogens from cerebrospinal fluid (CSF)were analyzed,DNA from patients’CSF were performed PCR amplification and sequencing with universal primers of bacterial 16S rRNA and fungal 28S rRNA, PCR detection results were compared with CSF culture during the same period.Results A total of 400 patients were with confirmed or suspected bacterial or fungal infection of CNS,132 of whom were with positive CSF culture.150 pathogenic isolates were detected,including 48 isolates of gram-positive bacteria,90 gram-negative bacteria,and 12 fungi;the top three isolated bacteria were Acinetobacter baumannii (n =32 ),coagulase negative staphylococcus (n=16)and Klebsiella pneumoniae (n=13);the most common fungus was Cryptococcus neoformans (n=8).CSF from 88 infected patients and 20 non-infected patients were selected for PCR amplification,the sensitive of PCR am-plification assay was higher than the culture method (35.23% [31/88]vs 28.41 %[25/88],χ2 =4.17,P <0.05).

Retentive bond strength of fiber-reinforced composite posts cemented with different surface treatments / 대한치과보철학회지

PURPOSE: This study will evaluate the effectiveness of various pretreatments when fiber-reinforced composite (FRC) post is bonded to endodontically treated tooth with resin cement. MATERIALS AND METHODS: Canal shaping of FRC post (DT Light post, Size 3, Bisco Inc., Schaumburg, IL, USA) was performed on endodontically treated premolars at 1.5 cm from CEJ. Samples were divided into 6 groups of surface treatment after conventional washing and drying to the canal. Total of 24 FRC posts were randomly divided into 6 groups of surface treatment as follows: Group C: control - no surface treatment, Group A: airborne-particle abrasion (Cojet sand, 3M ESPE), Group S: silanization (Bis-silane, Bisco Inc.), Group M: universal primer (Monobond-plus primer, Ivoclar Vivadent Inc.), Group AS: silanization after airborne-particle abrasion, Group AM: universal primer treatment after airborne-particle abrasion. Pretreated fiber posts were cemented with resin-based luting material and photo-polymerized and cut to the thickness of 1 mm. Push-out test using a universal testing machine was performed. Bonding failure strength of post dislodgement was measured and the type of bonding failure was classified. Data were analyzed with Kruskal-Wallis test and multiple comparison groups were performed using Tukey HSD value of rank test (alpha=0.05). RESULTS: Group AS showed significantly highest bonding strength. Group S, group AM, group A, and group M showed lower bonding strength in order. The control group showed the lowest bonding strength. CONCLUSION: Surface treatment with silane showed to be the most effective of the surface pretreatment methods for cementation of FRC post. Surface treatment with universal primer showed no significant difference compared with no surface treatment group as for bonding strength.

Effect of universal primer on shear bond strength between resin cement and restorative materials / 대한치과보철학회지

PURPOSE: The purpose of this study was to evaluate the difference in shear bonding strength between resin cements to dental materials when a universal primer (Monobond plus) was applied in place of a conventional primer. MATERIALS AND METHODS: Four groups of testing materials: gold alloy (Argedent Euro, n = 16), non precious metal (T-4, n = 20), zirconia (Cercon, n = 20) and glass ceramic (IPS e.max press, n = 20), were fabricated into discs, which were embedded in an acrylic resin matrix. The gold alloy specimens were airborne-particle abraded, 8 of the specimens were coated with Metal primer II, while the remaining 8 specimens were coated with Monobond plus. The non precious and zirconia specimen were airborne-particle abraded then, the control group received Alloy primer coating, while the other was coated with Monobond plus. Glass ceramic specimens were etched. 10 specimens were coated with Monobond-S and the remaining specimens were coated using Monobond plus. On top of the surface, Multilink N was polymerized in a disc shape. All of the specimens were thermal cycled before the shear bonding strength was measured. Statistical analysis was done with Two sample t-test or Mann-Whitney U test (alpha=.05). RESULTS: There were no significant differences in bonding strength depending on the type of primer used in the gold alloy and glass ceramic groups (P>.05), however, the bonding strengths of resin cements to non precious metal and zirconia groups, were significantly higher when the alloy primer was used (P<.05). CONCLUSION: Within the limitations of this study, improvement of universal primers which can be applied to all types of restorations is recommended to precious metals and zirconia ceramics. But, the bond strengths of non precious metals and zirconia ceramics were significantly lower when compared to a 10-MDP primer. More research is needed to apply universal primers to all types of restorations.

A UNIVERSAL PRIMER U2 LABELING METHOD FOR MICROARRAY ANALYSIS / 解剖学报

Objective To report a new method of fluorescent labeling technique in microarray studies: universal primer U2 labeling( UPL). The efficiency was compared of the UPL with that of random primer, restriction display labeling method and the reverse transcription coupled random primer spiking labeling method(RT-PSL). Methods Influenza viral RNA was labeled with both UPL and the conventional random primer labeling method as well as two other more laborious labeling methods( RD-direct and RD-incorporate), and hybridized with influenza virus oligonucleotide microarrays. The signals extracted from the microarrays were analyzed using SPSS 10.0 software. Results The fluorescent intensity, signal-to-noise ration(SNR), true positive ratio(TPR) of probes and labeling reproducibility of UPL were demonstrated to be higher than those of the Random primer approaches.Conclusion These results established that UPL is a valid new labeling protocol, which may have wide applications in the research and development of the microarray technology.

Development and optimization of a fluorescent differential display PCR system for studying bovine embryo development in vitro

Differential display is a widely used methodology to identify genes that are differentially expressed in biological samples. We developed a new protocol for the amplification and recovery of differentially expressed genes from extremely small initial amounts of RNA (10 to 25 pg mRNA) from preimplantation bovine embryos. The cDNAs generated with an anchor primer, associated with a universal sequence, were amplified with an arbitrary primer and a single fluorescently labeled primer. Amplification products were easily visualized with a fluorescence scanner, without the need for radioisotopes. Nineteen isolated fragments were cloned and sequenced, confirming the expected primer sequences and allowing the recognition and identification of gene transcripts involved in bovine embryonic physiology.

Diagnosis of Bacteremia by Universal Primer of Eubacteria / 대한임상병리학회지

BACKGROUND: The prompt detection of bacteremia continues to be one of the most important responsibilities of clinical microbiology. But clinical diagnosis of bacteremia remains difficult, particularly in the neonates and young children. And fastidious bacteria with specific growth requirements or bacteria requiring longer incubation period are apt to be negative results in blood cultures. Therefore, polymerase chain reaction (PCR) amplification which targets the highly conserved DNA sequences found in all eubacteria would permit fast and sensitive determination of the presence of bacteria in blood. METHODS: A primer pair (DG74, RW01) for highly conserved regions of bacterial DNA encoding 16S ribosomal RNA (rRNA) was utilized for PCR amplification. PCR results were compaired with blood cultures and PCR products were digested with SmaI restriction enzyme for cutting of recognition site. RESULTS: Among 44 blood specimens which organisms were isolated by blood culture, 41 samples were positive for PCR, and 3 samples which C. albicans, P. aeruginosa, and gram- positive bacillus isolated were negative. No signal was observed when blood obtained from person without clinical sign and or symptoms of bacteremia. All 41 PCR products (371 bp) were cutted in two DNA fragments (161 bp, 210 bp) by SmaI enzyme. CONCLUSIONS: We concluded that a single primer pair designed to anneal to a highly conserved region of bacterial DNA can amplify DNA specimens from different bacteria, while not amplifying human DNA. Because of early detection, molecular trial of patients with signs and symptoms of possible bacterial infection will decrease morbidity and mortality with bacteremia, this approach may make it possible to identify new, nonculturable bacterial pathogens. Furthermore, if we use the specific primers for gram positive or negative bacteria in PCR method, it would be a more useful diagnostic tool for the clinicians.