PCR is a powerful tool for the amplification of genetic sequences. It has been widely applied in molecular biology. It is generally used to amplify short segments (several hundreds basepairs to several kilobasepairs). It is difficult to amplify a long DNA segment. Based on the sequenced genes, it is known that most intact genes are very long. And intact gene is very important for the gene to express specially and effectively. Long PCR is a very useful tool to amplify intact genes for constructing special expression vectors. We have tried several chemicals to optimize long PCR system and found betaine was the best. Betaine, as an amino acid analogue with small tetraalkylammonium ions, could remarkably improve the amplification of long targets from the plant genome. The suitable concentration of betaine was between 1.0 mol/L and 2.5mol/L. We could effectively amplify a 9 kb DNA segment from maize genome DNA and a 16 kb DNA segment from plasmid. It was shown that different primers and different targets (different GC content) needed different concentrations of betaine. Betaine can reduce or eliminate non-special amplification. In the meantime we tried other additive chemicals, such as DMSO, glycerin, formamide. They were no notable results in long PCR.
Betaine ; pharmacology ; Polymerase Chain Reaction ; methods

OBJECTIVETo develop an one-tube multiplex RT-PCR assay for simultaneous detection of six human coronaviruses (HCoVs).

METHODSGenbank sequences of six human coronaviruses, including HCoV-NL63, HCoV-229E, SARS-CoV, HCoV-OC43, MERS-CoV, and HCoV-HKU1, were included as reference sequences. Primers were designed based on multiple alignment of reference sequences, targeting the conserved regions of each species of HcoV. Virus strains and nucleic acid preserved in our lab were used as template in developing this automatic-electrophoresis-based one-tube multiplex RT-PCR assay. Detection limits and reproducibility were also evaluated with these templates. Samples with infection of other respiratory viruses preserved in our lab were used to evaluate specificity of this assay. Finally, we tested this assay with 140 clinical samples that were validated by real-time PCR in parallel.

RESULTSThis automatic-electrophoresis-based multiplex RT-PCR assay was able to detect six human coronaviruses simultaneously. All positive samples in this study were detected with at least one specific fragment of anticipated length (195, 304, 332, 378, 415, 442 bp) . No fragment was detected in negative controls. Detection limits of 1.0×10(1-1.0)×10(2) copies/µl were achieved in tests of single virus. No cross reaction was observed with other respiratory viruses. This multiplex RT-PCR assay showed the same sensitivity and specificity to that of individual real-time RT-PCR validated with clinical samples. Both methods detected 28 positive samples (20%) .

CONCLUSIONSSix HCoVs can be detected in one tube by this novel multiplex RT-PCR assay with high sensitivity and specificity.

An improved method for extracting high quality and quantity RNA from a jelly mushroom and a dimorphic fungus—Tremella fuciformis which is especially rich in polysaccharides, is described. RNA was extracted from T. fuciformis mycelium M1332 and its parental monokaryotic yeast-like cells Y13 and Y32. The A260/280 and A260/230 ratios were both approximately 2, and the RNA integrity number was larger than 8.9. The yields of RNA were between 108 and 213 µg/g fresh wt. Downstream molecular applications including reverse transcriptional PCR and quantitative real-time PCR were also performed. This protocol is reliable and may be widely applicable for total RNA extraction from other jelly mushrooms or filamentous fungi rich in polysaccharides.
Agaricales* ; Fungi ; Humans ; Methods* ; Mycelium ; Parents ; Polymerase Chain Reaction ; Polysaccharides* ; Real-Time Polymerase Chain Reaction ; RNA*

The AB 7300 Real-time PCR Systems in our hospital have been detected malfunction for three times in a short term. The detection indicated the major reason for the malfunction attributed to the fuse of the solder of the COVER at the same position with the serial number 4344490 D1. This article explicitly introduces how to detect and maintain this equipment. It serves as a good reference for colleagues. Meanwhile the author raises five issues for further exploration and discussion.
Equipment Failure Analysis ; Maintenance ; methods ; Polymerase Chain Reaction ; instrumentation ; methods

Background: Prenatal diagnosis has become a standard part of obstetrics care. Genetic diagnoses are established prenatally through the sampling of fetal genetic material. So that the presence of fetal DNA in maternal plasma has led to exciting possibilities of non-invasive prenatal diagnosis. Objectives:In order to provide a reliable non-invasive method for diagnostic of the sex linked disorders. Subjects and method: Fetal gender was determined in 10 pregnant women in which 6 male fetus and 4 female fetus. They are between 34 and 36 weeks of gestation using DNA extracted from 1.6ml of each maternal serum. Maternal serum was put into vacutainer SST before delivery while two pregnant women were implemented by caesarean section at Hanoi Hospital for Obstetricts and Gynecology. Results:The 198bp SRY gene-specific sequence on Y chromosome, 261 bp ATL1 gene specific sequence on X chromosome were amplified in nested PCR. The results were confirmed by examination of newborn child after delivery.The mean level of using DNA extracted from maternal serum was 8.73 \xb1 2.36 ng/\xb5l. The mean level extracted fromperipheral blood was 66.2 \xb1 7.07 ng/\xb5l. Conclusion: The 198 bpSRY specific sequence we detected in 6 serum sample from pregnant women with male fetus. In the remaining cases bearing female foetuses only the 261 bp ATL1 gene sequence was detected. The result was completely concordant with the examination of the newborn child after delivery.
Serum/ immunology ; Prenatal Diagnosis/ methods ; Polymerase Chain Reaction/ methods

The theory of fluorescence resonance energy transfer (FRET) and methods of fluorescence detection in fluorescent-quantitative polymerase chain reaction (FQ-PCR) are introduced in this article. Applications of FRET in fluorescence detection of PCR are emphatically discussed, and FRET research progress and future trends are pointed out too.
Fluorescence Resonance Energy Transfer ; methods ; trends ; Polymerase Chain Reaction ; methods

OBJECTIVETo apply the single cell nested multiplex polymerase chain reaction (PCR) to HLA typing, and analyze the influence factors on the amplification results.

METHODSSingle cell DNA templates were prepared with different methods. The exon 2, 3 and intron 2 of HLA-A, B, and exon 2 of DRBI were amplified using multiplex PCR. The second round of SSP-PCR HLA typing was carried out according to the large scale routine HLA typing results.

RESULTSEnzyme lysis method was the most efficient procedure for preparing the single cell DNA template, with a success rate (SR) of 93.3%, while the SRs of alkali lysis and freezing-thaw lysis methods were 83.3% and 73.3%, respectively. The second round amplification using enzyme lysis and SSP-PCR in 20 samples obtained a 95% success rate and a 15% allele drop out rate. The time for performing the whole procedure was less than 6 hours.

CONCLUSIONThe modified nested multiplex PCR technique is efficient for single cell HLA typing and might be applied to clinical preimplantation genetic diagnosis.

The CO I gene sequences of Qianghuoyu, Pachytriton labiatus and Gehyra mutilata were achieved by PCR amplification and bi-directional sequencing. Furthermore, a pair of specific primers SJYW1 and SJYW2 in the non-conservative district were designed through sequence alignment. The PCR reaction condition was established by changing the annealing temperature and cycle numbers. The results showed that 350 bp DNA fragment was amplified from Qianghuoyu in PCR with annealed temperature at 54 °C and the cycle number was 25 cycles, whereas not any DNA fragment was amplified from P. labiatus and G. mutilata under the same reaction condition. This method is well-performed in the identification of Qianghuoyu for its excellent specificity and repeatability.
Animals ; Drug Contamination ; Medicine, Tibetan Traditional ; Polymerase Chain Reaction ; methods

OBJECTIVETo establish a specific TaqMan-based Real-time PCR assay for the detection of hepatitis A virus in serum samples.

METHODSAccording to the references, primers-probe sets which were located in 5'-NCR, the most conservative part of HAV genome were designed and therefore we established a TaqMan real-time RT-PCR assay with great performance of specificity, sensitivity and reproducibility. And then it was used in the detection of HAV RNA in serum from HAV patients.

RESULTSThe HAV Real-time RT-PCR assay established in this study were able to detect HAV RNA and its detection limit ranged from 0.1CCID50/reaction to 0.01CCID50/reaction. When the detection of a same sample was repeated for three times, coefficients of varistion (CV) of intra- and inter-assay were calculated and they were all less than 2.0% and 2.6% respectively. Our data suggested that there were 5.18 x 10(2) - 4.93 x 10(7) RNA copies in 1 ml of the serum from acute HAV patients.

CONCLUSIONThe TaqMan-based Real-time PCR assay established in this study was specific and precise for the rapid detection of HAV RNA. It was applied successfully in the pathogen detection of clinical samples.

PURPOSE: The aim of this study was to evaluate the diagnostic value of a peptide nucleic acid (PNA)-mediated PCR clamping method for the detection of BRAFV600E mutations in fine needle aspiration cytology (FNAC). METHODS: One hundred sixty four patients underwent FNAC to evaluate BRAFV600E mutations between April 2011 and November 2011. Among them, forty-two patients were diagnosed with papillary thyroid carcinoma in a permanent pathologic specimen. A PNA-mediated PCR clamping method and a Dual-Priming Oligonucleotide (DPO)-based Real-time PCR method were used to detect the BRAFV600E mutation. We compared the result of mutation between the two methods. RESULTS: A BRAF mutation was found in 31 samples created by the PNA-mediated PCR clamping method, and in 28 samples in the DPO-based Real-time PCR method. The rate of BRAF mutation was 73.8% in association with the PNA-mediated PCR clamping method, and 66.7% in association with the DPO-based Real-time PCR method. There was no statistical differences between the two methods (P>0.05). CONCLUSION: The PNA-mediated PCR clamping method may be an alternative to the DPO-based Real-Time PCR method for detection of BRAF mutations in thyroid nodules.
Biopsy, Fine-Needle* ; Constriction* ; Humans ; Methods* ; Polymerase Chain Reaction* ; Real-Time Polymerase Chain Reaction ; Thyroid Neoplasms ; Thyroid Nodule