Clinical application of colony PCR in rapid detection of pathogenic fungi causing tinea capitis / 中华皮肤科杂志

Objective To evaluate the reliability and clinical practicality of colony PCR in rapid detection of pathogenic fungi causing tinea capitis.Methods Totally,17 children with tinea capitis were enrolled from the Department of Dermatology of Wuxi No.2 People's Hospital between January 2016 and March 2017.Colony PCR was performed to detect pathogenic fungi.The results of colony PCR were compared with those of routine PCR and morphological identification,so as to evaluate the reliability of colony PCR in the identification of pathogenic fungi causing tinea capitis.Results After clinical specimens (broken hairs and scales) from the 17 patients were subjected to fungal culture,the mycelia were collected and successfully amplified by colony PCR.The time of clony PCR (mean,3.82 ± 0.50 days) for DNA template preparation was short than that of traditional morphological identification (14 days).Based on the results of conventional PCR,the accuracy of colony PCR for fungal identification was 100％,which was superior to that of conventional PCR (88.2％).Conclusions Colony PCR can be applied to the clinical detection of pathogenic fungi causing tinea capitis at specy level,and is a kind of rapid,economic and reliable molecular detection technique.

Isolation and Identification of Novel IndigenousBacterial Strain as a Low Cost Pectinase Source

ABSTRACT The present study was concerned with the searching of novel bacterial cultures from different samples for the lab scale production of pectinase. Keeping in view the increasing demand of pectinase specially in Faisalabad, an industrial city of Pakistan, isolation of new hyper producer bacterial strains locally is an easy and cheap way of getting the desirable products at low cost. Therefore, isolation of new strains for industrial enzyme production has been and will be remained a part of research every time. This method alone can also provide raw material for further research such as enzyme engineering or molecular directed evolution. For the identification of hyper producer strain colony PCR was done for 16S rRNA analysis. Reason to use the 16S rRNA for identification purpose is that the gene is fairly short and can be amplified quickly and easily. The bacterial isolate (sources of pectinase enzyme) was identified based on PCR amplification of 16S rRNA and for this purpose the amplified product was run in agarose gel against a known species of Bacillus licheniformis. The 16S rRNA sequencing confirmed the Bacillus status of the strain.

Knockout confirmation for Hurries: rapid genotype identification of Trypanosoma cruzi transfectants by polymerase chain reaction directly from liquid culture

Gene knockout is a widely used approach to evaluate loss-of-function phenotypes and it can be facilitated by the incorporation of a DNA cassette having a drug-selectable marker. Confirmation of the correct knockout cassette insertion is an important step in gene removal validation and has generally been performed by polymerase chain reaction (PCR) assays following a time-consuming DNA extraction step. Here, we show a rapid procedure for the identification of Trypanosoma cruzi transfectants by PCR directly from liquid culture - without prior DNA extraction. This simple approach enabled us to generate PCR amplifications from different cultures varying from 106-108 cells/mL. We also show that it is possible to combine different primer pairs in a multiplex detection reaction and even to achieve knockout confirmation with an extremely simple interpretation of a real-time PCR result. Using the “culture PCR” approach, we show for the first time that we can assess different DNA sequence combinations by PCR directly from liquid culture, saving time in several tasks for T. cruzi genotype interrogation.

A simple negative selection method to identify adenovirus recombinants using colony PCR

Background: The AdEasy system is a fast-track system for generating recombinant adenoviruses using the efficient homologous recombination machinery between shuttle and adenovirus backbone plasmids in Escherichia coli BJ5183 cells. The key step is homologous recombination in BJ5183 cells, which is driven by RecA activity. However, culture time is stringently limited to reduce the damage to recombinant plasmids by RecA activity. Therefore, rapid identification of recombinant adenoviruses within the limited time-period is critical. Results: We developed a simple negative selection method to identify recombinant adenoviruses using colony PCR, which improves the efficiency of adenovirus recombination screening and packaging. Conclusions: The negative selection method to identify AdEasy adenovirus recombinants by colony PCR can identify the recombined colony within a short time-period, and maximally avoid damage to the recombinant plasmid by limiting recombination time, resulting in improved adenovirus packaging.

A most effective method for selecting a broad range of short and medium-chain-length polyhidroxyalcanoate producing microorganisms

A molecular approach was used for selecting polyhydroxyalcanoate (PHA)-accumulating potential Gram-negative bacteria from different genera by colony polymerase chain reaction (PCR). Three degenerate primers were designed for amplifying a fragment from PHA synthase gene (phaC) (Class I), phaC1 and phaC2 (Class II) genes for detecting PHA-producing bacteria. Thirty-four out of 55 bacterial strains from the old collection selected using Sudan black B staining were phaC+. PCR was used for directly selecting 35 new collection bacterial strains; these strains were phaC+ and their ability to produce PHA was confirmed by Sudan black B staining. Four specific primers were designed on genes of Class II PHA biosynthesis operon. These primers were used for evaluating 9 strains from the old phaC+ collection; 6 showed Class II PHA synthase organisation. 34 from the old and new bacterial isolation were characterised by 16S ribosomal gene (16S rDNA) gene partial sequencing. The tool proposed here can be used for better directing PHA production based on PHA biosynthesis genes and bacterial genera. Class I or II phaC genes were detected in 9 different genera and were able to infer the type of polymer produced.

The Application of Colony PCR in the Molecular Biological Analysis of Malassezia Yeasts / 대한의진균학회지

BACKGROUND: Malassezia yeasts are lipophilic fungi that are found in 75~80% of healthy adults. The yeasts are known to be associated with pityriasis versicolor, seborrheic dermatitis, Malassezia folliculitis, and recently its pathogenicity is being expanded to other various skin disorders, such as atopic dermatitis and acne vulgaris. Recently, various molecular biological techniques are being preferred over morphological analysis. In order to perform a DNA-based diagnostic test, availability of a simple, rapid, and reliable DNA extraction protocol is essential. OBJECTIVE: We sought to implement novel molecular biology technique, namely colony PCR method using microwave as the easiest way to amplification of Malassezia target DNA, and assess its clinical applicability. METHODS: Instead of using templates of purified genomic DNA, we performed the PCR directly from Malassezia colonies. A fresh yeast colony transferred to the bottom of a microcentrifuge tube and microwaved for 1 min three times in the presence of a pyrex beaker containing 50 ml of sterile water to dissipate excess heat. Following this microwave lysis, PCR-reaction mixture was added directly to the microcentrifuge tube. Two DNA extraction methods (boiling method, glass beads method) were used for comparing the sensitivity and effectiveness with the colony PCR method. All reactions were performed using the primers 26S and ITS1 complementary to the rDNA region. Results 1. As a result of gel electrophoresis, we recognized expected PCR products (approximately 580 bp for 26S rDNA and 250~320 bp for ITS1) from both colony PCR method and two DNA extraction methods (boiling method, glass beads method). 2. As a result of measuring nucleic acid level with the spectrophotometer, colony PCR disregarding DNA extraction process shows relatively similar PCR efficacy compared with the boiling and glass beads method. And there is no significant difference among those methods statistically (p>0.001). 3. In conducting the PCR method, boiling method required approximately 400 minutes, and glass beads method required approximately 360 minutes, respectively. As contrasted with two methods, colony PCR method required approximately 150 minutes, and could be capable of saving time. In addithion, colony PCR had an economic efficiency comparing with boiling method and glass beads methods. CONCLUSIONS: All these findings suggest that directly application of the Malassezia yeasts obtained from culture colony for PCR reaction is a fast, reliable, cost-effective and simple method for performing any PCR-based protocol including diagnostic tests.

Distribution and Study on Single Nucleotide Polymorphism (SNP) of spvR Gene in Korean Isolates of Salmonella / 감염과화학요법

BACKGROUND: The Salmonella virulence plasmid (spv) genes (spvR, A, B, C and D) on the large virulence plasmids of pathogenic Salmonella serotypes can replace the virulence of the whole plasmid. Recently, virulence plasmid-negative pathogenic Salmonella isolates were isolated. However, positive rates of spv genes among Korean Salmonella serotypes have been obscure. spv genes are conserved in compared to other virulence genes but there are single nucleotide polymorphisms (SNPs) conserved in only certain serotype. Such SNPs are useful for differentiation and understanding evolution of certain serotypes. MATERIALS AND METHODS: Salmonella serotypes isolated from live stocks [Salmonella typhimurium (ST, 26), S. enteritidis (SE, 10), S. gallinarum (SG, 40) and S. pullorum (SP, 53)] were used for colony-PCR. A primer set covering single nucleotide polymorphism (SNP) at 625th nucleotide of spvR was designed. The nucleotide sequences of amplicons were determined by cyclic sequencing method and RFLP was performed by using MseI. RESULTS: All isolates of SE, SG and SP, including four plasmid-negative isolates, showed specific amplicons but not all of ST (19/26, 73%) were positive to spvR. Based on the nucleotide sequence of 625th nucleotide and PCR-RFLP, SE, SG and SP [A(625)] and ST [G(625)] could be differentiated. CONCLUSION: spvR can be used as a molecular marker to detect virulent SE, SG, SP and the SNP may be useful for differentiation of SE, SG, SP and ST. According to the SNP study SE may be evolutionarily closer to SG and SP than ST.

Distribution and Study on Single Nucleotide Polymorphism (SNP) of spvR Gene in Korean Isolates of Salmonella / 감염과화학요법

BACKGROUND: The Salmonella virulence plasmid (spv) genes (spvR, A, B, C and D) on the large virulence plasmids of pathogenic Salmonella serotypes can replace the virulence of the whole plasmid. Recently, virulence plasmid-negative pathogenic Salmonella isolates were isolated. However, positive rates of spv genes among Korean Salmonella serotypes have been obscure. spv genes are conserved in compared to other virulence genes but there are single nucleotide polymorphisms (SNPs) conserved in only certain serotype. Such SNPs are useful for differentiation and understanding evolution of certain serotypes. MATERIALS AND METHODS: Salmonella serotypes isolated from live stocks [Salmonella typhimurium (ST, 26), S. enteritidis (SE, 10), S. gallinarum (SG, 40) and S. pullorum (SP, 53)] were used for colony-PCR. A primer set covering single nucleotide polymorphism (SNP) at 625th nucleotide of spvR was designed. The nucleotide sequences of amplicons were determined by cyclic sequencing method and RFLP was performed by using MseI. RESULTS: All isolates of SE, SG and SP, including four plasmid-negative isolates, showed specific amplicons but not all of ST (19/26, 73%) were positive to spvR. Based on the nucleotide sequence of 625th nucleotide and PCR-RFLP, SE, SG and SP [A(625)] and ST [G(625)] could be differentiated. CONCLUSION: spvR can be used as a molecular marker to detect virulent SE, SG, SP and the SNP may be useful for differentiation of SE, SG, SP and ST. According to the SNP study SE may be evolutionarily closer to SG and SP than ST.

False positives resulted from colony PCR in rapid identification of recombinant clones from phage antibody library / 解放军医学杂志

Objective To evaluate the reliability of colony PCR in identifying the recombinant clones selected from phage antibody libraries. Methods The digested pComb3 vector and Lc fragments, Lc library plasmid, and Fd fragments were ligated successively. The ligation product was transformed into Escherichia Coli strain XL 1-blue bacilli by electroporation and thus murine Fab phage antibody library was constructed.The transformed recombinant clones selected randomly from libraries were identified simultaneously by colony PCR, plasmid PCR and restriction enzyme digestion. The identification consistency was analyzed. The interference was excluded by control PCR using the relevant constituents as template. Results The educed library content of Lc library was 1.175?10 6 CFU and the content of Fab antibody library was 1.02?10 6 CFU. Different recombinant percentages were obtained through three different identification methods (the Lc positive recombinant percentages by colony PCR, plasmid PCR and enzyme digestion were 100%, 78% and 78%, respectively; the Fd positive recombinant percentages by three methods were 90%, 66% and 66%, respectively; the dual positive recombinant (Fd and Lc insert simultaneously) percentages by three methods were 90%, 50% and 50%, respectively). A high frequency of false-positives appeared in colony PCR identification. Nonspecific amplification of control PCR was presumably induced by some intracellular components in XL 1-blue bacilli. Conclusion The identification of the recombinant clones selected from phage antibody libraries by colony PCR remains ambiguous. So it is our assertion that the traditional identification methods such as plasmid PCR or enzyme digestion are more accurate and will less false positive results.