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.

Coronavirus ; Humans ; Multiplex Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; methods ; Reproducibility of Results ; Sensitivity and Specificity

OBJECTIVETo explore the value of multiplex ligation-dependent probe amplification (MLPA) for rapid detection of aneuploidies and structural chromosomal abnormalities during prenatal diagnosis.

METHODSTwo hundred and eight six amniotic fluid samples were analyzed with both MLPA and conventional karyotyping. Structural abnormalities were verified with array comparative genomic hybridization.

RESULTSTen cases of trisomy 21, 2 cases of trisomy 18, 1 case of trisomy 13, 1 case of mosaic trisomy 21, 1 case of 45,X, 1 case of large deletion of Xp, 1 case of trisomy 18p and 1 case of large deletion of 18p and 18q were identified. The same results were derived by both MLPA and conventional karyotyping. Structural abnormalities were verified by array comparative genomic hybridization (aCGH) with 100% accuracy.

CONCLUSIONIn addition to aneuploidies, MLPA can rapidly identify large deletions and duplications of chromosomes 21, 18, 13, X and Y. MLPA is supplementary to conventional karyotyping for identification of such chromosomal abnormalities prenatal diagnosis.

Adult ; Aneuploidy ; Female ; Humans ; Multiplex Polymerase Chain Reaction ; methods ; Pregnancy ; Prenatal Diagnosis ; methods ; Young Adult

BACKGROUND: The aim of this study was to investigate the status of BRCA1/2 genetic testing practices in Korea in 2014. METHODS: A structured questionnaire was provided to the specialist in charge of BRCA1/2 genetic testing via e-mail between 28 July and 10 August 2015. A total of 11 genetic testing professionals from 14 organizations responded to the survey that asked about the status of BRCA1/2 genetic testing in the year 2014. RESULTS: The average number of BRCA1/2 genetic tests executed was 192; 6 organizations had executed less than 100 tests, and 5 organizations had conducted more than 100 tests. The primary testing method used was Sanger sequencing (100%), and 2 institutes performed multiplex ligation-dependent probe amplification (MLPA). The analysis software differed across the various organizations, with Sequencher (81.81%), Seqscape (27.27%), and Codoncode Aligner (9.09%) reported as utilized. We found that the guidelines for the interpretation of the genetic tests were different at each institution. CONCLUSIONS: Although this study only examined the status of the 2014 BRCA1/2 genetic testing practices of 11 institutions, it illustrates the necessity for standardized genetic testing or interpretation guidelines in Korea.
Academies and Institutes ; Electronic Mail ; Genetic Testing* ; Korea* ; Methods ; Multiplex Polymerase Chain Reaction ; Specialization ; Surveys and Questionnaires

OBJECTIVE: To develop a five fluorescence-labeled multiplex amplification system for 15 loci and study genetic polymorphism in Xinjiang Uygur population. METHODS: The STR loci were screened. The alleles were named according to the number of repeats by sequencing. The sensitivity, species specificity, identity and stability of the five fluorescence-labeled multiplex amplification system for the 15 loci were all tested. Then, the genetic polymorphism was analyzed in Xinjiang Uygur population and compared with other ethnic groups including Xizang Tibetan, Xiuyan Manchu, and Guangzhou Han population. RESULTS: The 15 loci multiplex amplification system was established. The sensitivity was 0.3 ng with good species specificity, identity and stability. The distributions of genotype for 13 STR loci in Uygur population were in accordance with Hardy-Weinberg equilibrium with no genetic linkage between these loci. Most loci showed statistically significant among different populations. CONCLUSION The established system has application value in forensic evidence. The 13 STR loci in Uygur population have
Alleles ; Ethnicity/genetics* ; Gene Frequency ; Genetic Linkage ; Genotype ; Humans ; Multiplex Polymerase Chain Reaction/methods* ; Polymorphism, Genetic

OBJECTIVE: To establish the rapid PCR amplification program and system and to verify the technical indexes. METHODS: PCR multiplex and capillary electrophoresis detection of 24 autosomal STR loci and one Y-STR loci using the 6-color fluorescence marking technology, as well as A melogenin and Y-InDel. Meanwhile, sensitivity, specificity, identity, stability, mixing and a batch of sample tests were investigated, and the genotype of various routine samples and degraded, exfoliated cell samples were observed. RESULTS: The sensitivity of the system was 0.062 5 ng. In addition, the genotype could be detected accurately only around 65 min via rapid amplification. The species-specificity was high and the genotyping of all kinds of dry blood specimens of filter paper and mixed, degraded, exfoliated cell samples were accurate. CONCLUSION The rapid amplification system can significantly improve the detection rate, and obtain accurate and stable genotyping results, which may be important implications for the establishment of STR database and study on population genetics and forensic identification.
Electrophoresis, Capillary ; Fluorescence ; Genetics, Population ; Genotype ; Humans ; Microsatellite Repeats ; Multiplex Polymerase Chain Reaction/methods* ; Sensitivity and Specificity

OBJECTIVES: To develop a multiplex PCR amplification system (EX20+30Y for short) of 19 autosomes, 30 Y-STR loci plus the gender indicator, and evaluate its forensic application value. METHODS: With the six-color fluorescence labeling technology, a multiplex amplification system of 19 autosomal STR loci and 30 Y-STR loci plus the gender indicator was constructed. Blood samples from 210 unrelated individuals, 69 daily case samples and standard samples 9948 and 9947A were collected for loci detection and analysis. The EX20+30Y multiplex amplification system was evaluated by its sensitivity, mixed sample detection ability, species specificity, balance, direct amplification ability, sample applicability and anti-inhibition ability. RESULTS: Multiplex amplification of blood samples from 210 unrelated individuals by the detection system obtained accurate genotyping results. The detection sensitivity of standard samples was 0.125 ng and the species specificity was high. The 69 samples from daily cases were genotyped correctly. Moreover, standard sample 9948 could be accurately genotyped even if the samples contained a certain concentration of inhibitors. CONCLUSIONS The multiplex amplification system established in this study can conduct combined examination of 19 autosomes, 30 Y-STR loci plus the gender indicator with accurate genotyping and high sensitivity. It has a good forensic application prospect.
Chromosomes, Human, Y/genetics* ; DNA Fingerprinting/methods* ; Forensic Medicine/methods* ; Humans ; Microsatellite Repeats ; Multiplex Polymerase Chain Reaction ; Species Specificity

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.
Cryptosporidium parvum* ; Cryptosporidium* ; Cyclospora* ; Diarrhea* ; Genes, rRNA ; Giardia lamblia* ; Giardia* ; Glutamate Dehydrogenase ; Humans ; Methods ; Multiplex Polymerase Chain Reaction ; Oocysts ; Parasites ; Polymerase Chain Reaction* ; RNA, Ribosomal, 18S

OBJECTIVEThis study is aimed to develop a two-tube melting curve-based multiplex real time PCR assay (MCMRT-PCR) for the simultaneous detection of six common foodborne pathogenic bacteria (diarrhoeagenic Escherichia coli, Salmonella, and Shigella in tube 1, Staphylococcus aureus, Vibrio parahaemolyticus, and Listeria monocytogenes in tube 2).

METHODSA two-tube MCMRT-PCR assay was performed on 7900HT Fast Real-Time PCR System (Applied Biosystems, USA). Amplification by PCR was optimized to obtain high efficiency. The sensitivity and specificity of assays were investigated.

RESULTSThe detection limit of optimized MCMRT-PCR assay was 3.9×102 CFU/mL for S. aureus, 4.4×102 CFU/mL for L. monocytogenes, 3.0×102 CFU/mL for Salmonella, 2.5×102 CFU/mL for Shigella, 2.1×102 CFU/mL for V. parahaemolyticus, and 1.2×102 CFU/mL for E. coli. The feasibility of MCMRT-PCR was further evaluated using artificially contaminated milk, the sensitivity was at the level of 105 CFU/mL.

CONCLUSIONA two-tube MCMRT-PCR assay using six primer sets was developed for detection of multiple pathogens. Our findings demonstrates that the proposed two-tube assay is reliable, useful and rapid for simultaneous detection of six foodborne pathogenic bacteria with an intended application in provincial Centers for Diseases Control and Prevention (CDC).

Animals ; Bacteria ; genetics ; isolation & purification ; Food Microbiology ; methods ; Milk ; microbiology ; Multiplex Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Sensitivity and Specificity

A multiplex PCR method was developed to detect the main pathogens of Qinghai Tibetan sheep endometritis. First, the genomes of five standard bacterial strains were extracted and specific primers were selected; the multiplex PCR method was established by using the genome of the standard strain as a template. The samples were collected by sterile cotton swab from Tibetan sheep uterus, and then placed in LB medium and numbered. After 48 h, the genomes of cultured bacteria were extracted and detected by single PCR method, then the positive samples were recorded. The positive samples detected by single PCR were selected for multiplex PCR detection and recorded again. The coincidence rate between these two methods was calculated to measure the accuracy of multiplex PCR. In order to identify the species of the pathogen, 30 positive samples verified by single and multiplex PCR were randomly selected for bacterial isolation and identification. In the 600 samples, the infected ratio of Streptococcus agalactiae (GBS) was 47.33%, Escherichia coli 34.83%, Staphylococcus aureus 6.5%, Salmonella and Trueperella pyogenes were negatively detected. Among the positive samples detected by multiplex PCR, the positive ratio of GBS was 45.50%, E. coli 33.50%, S. aureus 6.5%. Comparison of two detection results, Multiplex PCR detection coincidence rate is more than 95%. The isolated pathogens were identified as E. coli, GBS and S. aureus, which was consistent with the results of two methods. The multiplex PCR method was successfully established and the main pathogens of endometritis in Qinghai Tibetan sheep were GBS, E. coli and S. aureus.
Animals ; Bacteria ; genetics ; isolation & purification ; Bacteriological Techniques ; methods ; Endometritis ; microbiology ; veterinary ; Female ; Multiplex Polymerase Chain Reaction ; standards ; Polymerase Chain Reaction ; veterinary ; Sensitivity and Specificity ; Sheep ; Sheep Diseases ; microbiology ; Tibet

OBJECTIVES: Studies on Clostridium difficile are rare in Korea. We investigated the epidemiological characteristics of C. difficile isolates from patients with C. difficile-associated disease (CDAD) in Korea. METHODS: Multiplex polymerase chain reaction was performed to detect the presence of tcdA and tcdB toxin genes. Antimicrobial susceptibility test was carried out by the disk-dilution method. C. difficile strains were subtyped by automated repetitive-element palindromic PCR (rep-PCR). RESULTS: Among patients with CDAD, 73 (25.8%), 32 (11.3%), 32 (11.3%), and 26 (9.2%) suffered from pneumonia, cancer or neoplasm, diabetes, and colitis, respectively. Of all stool samples, 43 samples (15.2%) were positive for C. difficile strains. We observed two expression patterns of toxin genes: tcdA+/tcdB+ (86% isolates) and tcdA−/tcdB+ (14% isolates), with all isolates expressing tcdB. Furthermore, some isolates were resistant to clindamycin (65%), ampicillin (56%), and cefazolin (40%), but all were susceptible to vancomycin and metronidazole. The tested samples were classified into diverse clusters using automated rep-PCR. CONCLUSION: Our findings revealed the characteristics and antibiotic resistance of C. difficile isolates from patients in Korea. The epidemiological data may provide valuable insight into development of treatment strategies for C. difficile infections in Korea.
Ampicillin ; Cefazolin ; Clindamycin ; Clostridium difficile* ; Clostridium* ; Colitis ; Drug Resistance, Microbial ; Humans ; Korea* ; Methods ; Metronidazole ; Multiplex Polymerase Chain Reaction ; Pneumonia ; Polymerase Chain Reaction ; Vancomycin