Duplex PCR for rapid detection of the drug-resistant genes KPC and NDM / 医学研究生学报

ObjectiveThe nucleic acid technology for detecting drug-resistant genes has become one of the powerful tools for monitoring and controlling the spreading of drug-resistant bacteria. This study was to establish a method for rapid detection of the drug-resistant genes KPC and NDM and provide some guidance in clinical drug use and monitoring the prevalence of drug-resistant bacteria in the hospital.MethodsAccording to the conserved regions of Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-β-lactamase (NDM), we designed the primers of duplex PCR, optimized the amplification system and established a method for simultaneous detection of the drug-resistant genes KPC and NDM. Then, we analyzed the sensitivity and specificity of the method and applied it to the detection of Pseudomonas aeruginosa and Klebsiella pneumoniae.ResultsThe sequences of KPC and NDM exhibited a 100% consistency with those of the original ones. Target fragments of the desired size of 151 bp were detected in the KPC-2 positive standard and Klebsiella pneumoniae ATCC BAA 1705 standard strains, and those of the desired size of 261 bp were observed in the NDM-2 positive standard strain and NDM-positive pneumococcal bacteria, neither with non-specific amplification. Sequencing of the PCR products showed a 100% consistency between the sequences of the products and those of the drug-resistant genes KPC-2 and NDM-1. The detectable limits of KPC and NDM for duplex PCR were 7×102 and 5×102 copies per reaction respectively. Drug-resistant genes were detected in 12 (92.3%) of the 13 carbapenems-resistant strains, including 10 KPC-positive (83.3%) and 2 NDM positive ones (16.7%), but neither KPC nor NDM in the other 10 carbapenems-sensitive strains. In the 13 strains of Pseudomonas aeruginosa, KPC was detected in 2 (33.3%) of the 6 carbapenems-resistant ones, but neither KPC nor NDM in the other 7.ConclusionThe duplex PCR method can be used for rapid and effective detection of the drug-resistance genes KPC and NDM, with the advantages of high sensitivity and specificity, and is therefore of great significance for guiding clinical drug use and monitoring the spreading of carbapenems-resistant bacteria in the hospital.

Development and Evaluation of a Duplex Real-Time PCR Assay With a Novel Internal Standard for Precise Quantification of Plasma DNA

BACKGROUND: Circulating levels of cell-free DNA increase in many pathologic conditions. However, notable discrepancies in the quantitative analysis of cell-free DNA from a large number of laboratories have become a considerable pitfall, hampering its clinical application. METHODS: We designed a novel recombinant DNA fragment that could be applied as an internal standard in a newly developed and validated duplex real-time PCR assay for the quantitative analysis of total cell-free plasma DNA, which was tested in 5,442 healthy adults and 200 trauma patients. RESULTS: Compared with two traditional methods, this novel assay showed a lower detection limit of 0.1 ng/mL, lower intra- and inter-assay CVs, and higher accuracy in the recovery test. The median plasma DNA concentration of healthy males (20.3 ng/mL, n=3,092) was significantly higher than that of healthy females (16.1 ng/mL, n=2,350) (Mann-Whitney two-sample rank sum test, P<0.0001). The reference intervals of plasma DNA concentration were 0-45.8 ng/mL and 0-52.5 ng/mL for healthy females and males, respectively. The plasma DNA concentrations of the majority of trauma patients (96%) were higher than the upper normal cutoff values and were closely related to the corresponding injury severity scores (R²=0.916, P<0.0001). CONCLUSIONS: This duplex real-time PCR assay with a new internal standard could eliminate variation and allow for more sensitive, repeatable, accurate, and stable quantitative measurements of plasma DNA, showing promising application in clinical diagnosis.

Development and application of duplex PCR for detection of H-1 and KRV strains / 中国比较医学杂志

Objective To develop a duplex PCR assay for detection of rat parvovirus H-1 and KRV and its application.Methods To design specific primers on the basis of H-1 ( NC_001358 ) and KRV ( U790330 ) genome sequences published in NCBI and establish a duplex PCR assay using H-1 and KRV DNA as templates.To verify the sensitivity and specificity of the method after optimizing PCR.The rats were infected by oral inoculation.The rats were divided into three groups:H-1 infection, KRV infection and mixed infection groups.To collect feces at the 4th, 6th, 8th and 10th days postinfection.Rats were euthanized on the 10th day and samples from heart, liver, spleen, lung, kidney and cecal contents were collected from each rat, then all the samples were screened with the duplex PCR.Results The 183 bp and 302 bp bands were amplified using H-1 and KRV as templates.The sensitivity test showed that the PCR method can detect as low as 3.8 pg/mL H-1 and 0.73 pg/mL KRV.There were no bands amplified when mouse minus virus, canine parvovirus and feline parvovirus were used as templates, showing that the specificity of the duplex PCR assay is very good. The nucleic acids of H-1 or KRV were detected in all rat feces on the 2th day postinfection and there was no obvious clinical symptoms in all the infected rats.The positive rates of H-1 were as follows:50%(4/8) heart tissues, 50%(4/8) liver tissues, 62.5%(5/8) spleen tissues, 50%(4/8) lung tissues, 37.5%(3/8) kidney tissues and 62.5%(5/8) cecum contents, and the positive rate of single infection group was higher than that of mixed infection group.The positive rates of KRV were as follows:0 (0/8) heart tissues, 25% (2/8) liver tissues, 87.5% (7/8) spleen tissues, 12.5% (1/8) lung tissues, 25%(2/8) kidney tissues and 62.5%(5/8) cecum contents, and the positive rate of mixed infection group was higher than that of single infection group.Conclusions The duplex PCR assay for H-1 and KRV established in this study can effectively detect H-1 or KRV infection in rat feces and other tissues, and can be used as an effective supplement to the national standard of lab animals.

Combination of duplex PCR technology with capillary electrophoresis is an alterna-tive method to validate the result of transcriptome sequencing / 军事医学

Objective To establish a method to validate the result of transcriptome sequencing using duplex PCR tech -nology combined with capillary electrophoresis .Methods According to a previous study on transcriptome sequencing , eight differentially expressed genes were chosen as target genes for examination .The mRNA expression level of these genes was detected using duplex PCR combined with agarose gel electrophoresis , duplex PCR combined with capillary electropho-resis and Q-PCR, respectively.Then, the verification efficiency of each method was evaluated carefully .Results The ver-ification efficiency of duplex PCR combined with agarose gel electrophoresis was 50%, while that of duplex PCR combined with capillary electrophoresis and Q-PCR was both 100%.Conclusion Combination of duplex PCR technology with capil-lary electrophoresis can be used as an alternative method to validate the results of transcriptome sequencing .

The Duplex PCR Detection of Transgenic Maize Transformed by Rice Rough Dwarf Virus Movement Protein Defective Gene / 中国生物工程杂志

A simple and effective method for the duplex PCR detection was developed by using sequences of exogenous gene(RDV MP-)and endogenous gene(Zein)as templates for PCR amplification.The results of routine PCR amplification for RDV MP-gene in transgenic maize suggested that RDV MP-gene can stably inheritate in transgenic plants and their progenies;The duplex PCR detection of all negative and part positive samples that obtained by routine PCR amplification confirmed that above negative results were exact,also showed that the quality of extracted DNA can meet the need of PCR amplification.The error ratio of negative samples was 1.4%.The method used in this study was simple and credible and can be used to detect transgenic plants and their products.

Cytogenetic diagnosis on single lymphocyte of DMD patient with exon 50 deletion by two-time duplex PCR / 第三军医大学学报

Objective To perform the cytogenetic diagnosis on single lymphocyte of DMD patient with dystrophin gene exon 50 deletion.Methods Single lymphocytes of a DMD patient with dystrophin gene exon 50 deletion and normal volunteers were picked out and prepared for two-time duplex PCR.Results The rate of precise positive was 92%,91% and 93% in specimens of the patient(SRY positive,exon 50 negative),the male volunteer(SRY positive,exon 50 positive)and the female volunteer(SRY negative,exon 50 positive),respectively.Conclusion Two-time duplex PCR is fit for the genetic diagnosis of single lymphocyte from DMD patient with dystrophin gene exon 50 deletion.