Construction of DNA ladder based on 16S rRNA gene of Bacillus subtilis using touchdown PCR technique

Aim@#DNA molecular size markers or DNA ladders play a vital role in molecular biology laboratories where DNA electrophoresis experiments are usually conducted. This study aimed to produce a 100 bp DNA ladder at laboratory scale, which could be applied to determine the size of DNA fragments in molecular biology experiments.@*Methodology and results@#In this study, 14 primers including 4 forwards and 10 reverses were designed based on the 16S rRNA gene sequence of Bacillus subtilis. These primers were able to amplify 10 DNA fragments with accurate sizes from 100 to 1000 bp. Furthermore, touchdown PCR was involved to maximize the specificity and yield of PCR products. Ten DNA fragments with the sizes including 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 bp were synthesized, and such bands were equivalent with commercial DNA ladders. Moreover, the quantity and quality of PCR products were measured using a nanodrop spectrophotometer. The optimal concentration ratios between such fragments (100- 1000 bp) were 800, 300, 150, 150, 500, 50, 50, 50, 50 and 50 (ng/µL), respectively. These ratios showed the clear and high resolution on 1.5% agarose gel. @*Conclusion, significance and impact of the study@#The results indicated that 16S rRNA gene of B. subtilis was a potential material for DNA ladder preparation due to the multiple copies number of this gene. Furthermore, in combination with touchdown PCR, the nonspecific bands were reduced, and the products could be used directly without the need of purification step.

Multiplex-Touchdown PCR to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the Major Causes of Traveler's Diarrhea

This study aimed to develop a multiplex-touchdown PCR method to simultaneously detect 3 species of protozoan parasites, i.e., Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the major causes of traveler’s diarrhea and are resistant to standard antimicrobial treatments. The target genes included the Cryptosporidium oocyst wall protein for C. parvum, Glutamate dehydrogenase for G. lamblia, and 18S ribosomal RNA (18S rRNA) for C. cayetanensis. The sizes of the amplified fragments were 555, 188, and 400 bps, respectively. The multiplex-touchdown PCR protocol using a primer mixture simultaneously detected protozoa in human stools, and the amplified gene was detected in >1×10³ oocysts for C. parvum, >1×10⁴ cysts for G. lamblia, and >1 copy of the 18S rRNA gene for C. cayetanensis. Taken together, our protocol convincingly demonstrated the ability to simultaneously detect C. parvum, G. lamblia, and C. cayetanenesis in stool samples.

Establishment and application of multiplex touchdown PCR for detection of extended-spectrum β-lactamases-producing Enterobacteriaceae and me-thicillin-resistant Staphylococcus aureus in bloodstream infection / 中国感染控制杂志

Objective To develop a multiplex touchdown PCR for simultaneous detection of extended-spectrum β-lactamases (ESBLs )-producing Enterobacteriaceae and methicillin-resistant Staphylococcus aureus (MRSA ). Methods Blood culture positive specimens from 102 hospitalized patients were collected between March 2013 and September 2014,four pairs of specific primers were designed based on SHV,TEM,and OXA genes of ESBLs-pro-ducing Enterobacteriaceae and MecA gene of MRSA,drug-resistant genes were amplified with single touchdown PCR and multiplex touchdown PCR, the results were compared with Kirby-Bauer disk diffusion method. Results Each single PCR amplified a specific band,four drug-resistant genes were also detected by multiplex touchdown PCR;the lower detection limits of multiplex touchdown PCR for DNA of MecA,SHV,TEM,and OXA were 4.37 ng,2.19 ng,4.53 ng,and 3.59 ng,respectively.Compared with Kirby-Bauer disk diffusion method, the overall sensitivity and specificity of multiplex touchdown PCR were 100.00% and 88.24% respectively,for ES-BLs were 100.00% and 87.23% respectively,for MRSA were both 100.00%.Conclusion A higher sensitivity and specificity multiple touchdown PCR assay has been developed,and it can be used in the rapid diagnosis and epidemi-ology investigation of bloodstream infection caused by ESBLs-producing Enterobacteriaceae and MRSA,and is help-ful for guiding antimicrobial use in clinic.

Evaluation of small-subunit rRNA touchdown polymerase chain reaction for direct detection of Entamoeba histolytica in human pus samples from patients with amoebic liver abscess.

Purpose: The aim of the present study was to evaluate the use of touchdown polymerase chain reaction (TD-PCR) for the detection of Entamoeba histolytica in liver pus samples obtained from patients with a clinical diagnosis of amoebic liver abscess (ALA) using small-subunit rRNA (SSU rRNA) as the target gene. Materials and Methods: Microscopic examination in vitro culture and serological test for the detection of E. histolytica in 67 pus samples obtained from ALA patients was performed. Molecular studies were carried out by both conventional PCR and TD-PCR targeting the SSU rRNA gene using the same sets of primers and the results were compared. Results: TD-PCR detected the presence of E. histolytica in 86.5% of the liver pus samples within 2.5 h as compared with 82.08% by conventional PCR within 3.5-4 h. Conclusion: TD-PCR assay may serve as a relatively better detection method for E. histolytica over conventional PCR with respect to the turnaround time, increased sensitivity, specificity and yield.

Polymorphism in CD14 promoter region in Han population in Chongqing / 第三军医大学学报

Objective To investigate the distribution of CD14C ( 260)T single nucleotide polymorphism (SNP) in the Han population in Chongqing and to test the reliability with this random sample in genetic research. Methods Single round Touchdown PCR (TD PCR) and restriction fragment length polymorphism (RFLP) method were used for genotyping in 110 people from the Han population in Chongqing. Results Frequencies of TT, CT, and CC genotypes were 43 6%, 39 1%, and 17 3%, respectively. The sample distribution was accorded with Hardy Weinberg principle. Conclusion CD14C( 260)T SNP was found in the Han population in Chongqing, and the distribution of alleles was significantly different from that in white population. The sample from an H W equilibrium population can be used in the association study of the systematic inflammatory reaction in the Han population in Chongqing.

Detection of Chlamydia pneumoniae by 'Touchdown' PCR / 대한임상병리학회지

BACKGROUND: Chlamydia pneumoniae has recently been established as an important cause of acute respiratory tract infections such as pneumonia and bronchitis in humans. We introduced a 'touchdown' PCR method for detection of C. pneumoniae from sputum. METHODS: A total of 474 patients with respiratory infection were enrolled in the study. The sputum samples were tested for C. pneumoniae by the 'touchdown' PCR and cultured for Chlamydia. The sputum samples were pretreated with 5% NaOH for mucolysis. In 'touchdown' PCR, the first round PCR amplified DNA from both C. pneumoniae and Chlamydia psittaci, while the second round specifically targeted C. pneumoniae, allowing the two species to be differentiated. RESULTS: The 'touchdown' PCR could detect 10-2 inclusion forming unit (IFU) in the 1st round and 10-3 IFU in the second round PCR. None of the C. trachomatis serovars, C. psittaci and other organisms tested was amplified. 'Touchdown' PCR detected C. pneumoniae DNA in 24 (5%) of the 474 sputum samples. Nine patients with C. pneumoniae had community acquired pneumonia. Another nine patients had pulmonary tuberculosis of which three had coexisting pneumonia. Two patients had lung cancer, another two had chronic bronchitis, one had pharyngitis, and one person was a normal healthy individual. CONCLUSIONS: The sputum preparation with 5% NaOH and the 'touchdown' PCR method are effective in the detection of C. pneumoniae. C. pneumoniae is one of the most common causative agents for pulmonary infection.