The value of multiplex PCR method based on capillary electrophoresis in detecting common serotypes of respiratory adenovirus infection in children / 中华实用儿科临床杂志

Objective:To establish a common method for detecting serotypes of respiratory adenovirus, and to detect the main serotypes of respiratory human adenovirus (HAdV) infection in children in Wenzhou area.Methods:A multiplex PCR method based on capillary electrophoresis was developed to detect 12 common serotypes of respiratory adenovirus.A total of 1 059 children with acute respiratory infection who were admitted to Yuying Children′s Hospital Affiliated to Wenzhou Medical University from January 2018 to December 2019 with positive infection of HAdV detected by the direct immunofluorescence method were recruited and retrospectively analyzed.Multiplex PCR was performed to determine 12 serotypes of respiratory adenovirus, including HAdV-1, 2, 3, 4, 5, 7, 14, 21, 37, 40, 41 and 55.Meanwhile, some samples were randomly selected to examine the consistency in the detection result by the first-generation sequencing method.Results:A total of 1 059 specimens of respiratory secretions with positive HAdV antigen were collected.Detected by multiplex PCR method, 947 cases (89.4%) were positive for 1 serotype, 13 cases (1.2%) were mixed infection with 2 serotypes, and 24 cases (2.3%) were negative.In addition, 75 cases(7.1%) were positive but could not be serotyped.Among the 947 children with the positive infection of a single serotype, 415 cases (43.8%) were HAdV-3 in subgroup B, 318 cases(33.6%) were HAdV-7, 12 cases (1.2%) were HAdV-55, 2 cases (0.2%) were HAdV-21, 108 cases (11.4%) were HAdV-2 in subgroup C, 70 cases (7.4%) were HAdV-1, 16 cases(1.7%) were HAdV-5, and 6 cases(0.6%) were HAdV-4 in subgroup E. HAdV-14, HAdV-37, HAdV-40 and HAdV-41 were not detected.A total of 51 positive samples of HAdV infection detected by multiplex PCR were randomly selected to compare with the detection result by the first-generation sequencing, which were all consistent.Conclusions:This study successfully established a multiplex PCR based on capillary electrophoresis in diagnosing common serotypes of respiratory adenovirus infection in children.HAdV-3, HAdV-7 of subgroup B and HAdV-2 and HAdV-1 of subgroup C were the main serotypes of respiratory adenovirus infection in children of Wenzhou area.HAdV-14, HAdV-37, HAdV-40 and HAdV- 41 were not detected.

Prueba de concepto de una pcr multiplex de primera generación (cualitativa) para detección del oncogén e7 de vphs de alto riesgo

Introducción: Infección persistente con el virus de papiloma humano de alto riesgo (VPH-AR) causa cáncer de cuello uterino (CCU). Existen ensayos moleculares para la detección y la genotipificación del gen L1 de VPH, sin embargo, L1 puede perderse durante la integración viral. La expresión e integración del oncogén E7 es fundamental para el desarrollo de CCU. Objetivo: Estandarizar una PCR multiplex (mPCR) del oncogén E7 (E7-mPCR) para genotipificación de los VPH-AR de mayor frecuencia en CCU (VPH-16, -18, -31, -33, -45 y -52). Métodos: Se obtuvieron cepillados cervicales de voluntarias y se analizaron amplificando por PCR el gen L1 con subsecuente hibridación reversa. Posteriormente, se escogieron 59 muestras positivas para VPH-AR y se analizaron por E7-mPCR. Resultados: Se evidenció una elevada concordancia entre los resultados del ensayo E7- mPCR y los de la PCR de L1 (concordancia observada de 95,1%, Kappa de Cohen = 0,88), encontrándose mayor número de infecciones por VPH- AR en el 15,8% con E7-mPCR. Conclusión: E7-mPCR es una herramienta diagnóstica con alta concordancia y económica que puede adaptarse a una plataforma de mayor complejidad para procesar y detectar mayor cantidad de muestras y genotipos de VPH-AR.

Introduction: The persistent infection of the high-risk Human Papiloma Virus (VPH-AR in Spanish) causes uterine cervix cancer (CCU in Spanish). There are molecular essays for detection and genotyping of gen L1 of VPH. However, L1 may get lost during the viral integration. The expression and integration of oncogene E7 is fundamental for the development of CCU. Objective: To standardize a multiplex PCR (mPCR) of oncogene E7 (E7-mPCR) for genotyping the VPH- AR of highest frequency in CCU (VPH-16, -18, -31, -33, -45 y -52). Method: We obtained cervix brushing simples from volunteers and we analyzed them by amplifying the L1 gene through PCR with a subsequent reverse hybridization. After that, we chose 59 positive VPH- AR samples and we analyzed them for E7-mPCR. Results: We found out a high concordance between the results of the essay E7-mPCR and those of L1 PC (Observed concordance was of 95.1%, Cohen's Kappa = 0.88), and we revealed a higher number of infections for VPH-AR in a 15.8% with E7-mPCR. Conclusion: E7-mPCR is an economic diagnostic tool with high concordance which can be adapted to a platform with more complexity to process and detect a higher number of samples and VPH-AR genotypes.

Introdução: a infecção persistente com o virus de papiloma humano de alto risco (HPV-AR) causa cáncer de colo do útero (CCU). Existem ensaios moleculares para detecção e para a genotipificação do gene L1 de HPV; contudo, L1 pode ser perdido durante a integrado viral. A expressão e integração do oncogênese E7 é fundamental para o desenvolvimento do CCU. Objetivo: padronizar uma PCR multiplex (mPCR) do oncogênese E7 (E7-mPCR) para genotipificação dos HPV-AR de maior frequência no CCU (HPV-16, -18, -31, -33, -45 e -52). Métodos: foram realizadas raspagens com escova cervical rodada em voluntárias e foram analisadas a partir da amplificação do gene L1 por PCR com subsequente hibridação inversa. Em seguida, foram escolhidas 59 amostras positivas para HPV-AR, as quais foram analisadas por E7-mPCR. Resultados: foi evidenciada elevada concordância entre os resultados do ensaio E7-mPCR e os da PCR de L1 (concordância observada de 95,1%, Kappa de Cohen = 0,88), encontrando-se maior número de infecções por HPV-AR em 15,8% com E7-mPCR. Conclusão: E7-mPCR é uma ferramenta diagnóstica com alta concordância e económica que pode ser adaptada a uma plataforma de maior complexidade para processar e detectar maior quantidade de amostras e genótipos de HPV-AR.

Development of A Multiplex PCR Method for Detecting Six Types of Viruses Causing Encephalitis / 现代检验医学杂志

Objective To develop a new mPCR method for rapid diagnosis of six types of encephalitis causing viruses of HS-VI,HSVII,VZV,EBV,EV71 and CMV.Methods Six pairs of specific primers for CMV,EV71,HSV I,VZV,EBV and HSV II were designed.The mPCR detection method was established and the sensitivity was detected.In order to verify the clinical application value of their multiplex PCR system,fifteen cerebrospinal fluid specimens of clinically suspected VE from the First Affiliated Hospital of Soochow University from 2014 to 2015 were examined by the mPCR method.Results The 6 pairs of primers did not interfere with each other,and the sensitivity of the mPCR system was over 103copies/μl.Among 15 cerebrospinal fluid specimens from patients with suspected viral encephalitis,six specimens(6/15,40%)were tested positive by the mPCR.Among them,HSV I was 5 and CMV was 1.Conclusion The mPCR method for detecting six types of en-cephalitis-associated virus at same time was established with high specificity,sensitivity and stability.

Establishment and application of a genotyping technique for detection of different DNA regions in Yersinia pestis using multiple polymerase chain reaction / 中华地方病学杂志

Objective By the method of multiple polymerase chain reaction (PCR),we intend to amplify different regions (DFR) of Yersinia pestis DNA,and to establish a multiple DFR genotyping technique for detection of Yersinia pestis.Methods According to the product size of 23 DFRs and pMT plasmid,24 primers were optimized and combined,then multiple primers in one PCR reaction system were added,and positive template DNA was amplified.Meanwhile,200 wild strain DNAs were amplified by multiple PCR and normal PCR,to verify the coincidence rate of the two methods.Results Totally 24 target segments were amplified through the positive DNA template.Through different permutation and combination,24 primers were optimized and combined into 9 groups.Totally 200 wild strain DNAs were used for verification,the coincidence rate of multiple PCR and normal PCR was 100％.Conclusions Multiple PCR is applicable and feasible for DFR genotyping of Yersinia pestis.It is an efficient,economic and high accuracy experimental method for large quantities of Yersinia pestis DFR genotyping.

Caracterización del gen mecA de Staphylococcus aureus resistentes a meticilina aislados de tres grupos poblacionales de la ciudad de Medellín / Characterization of mecA gene of methicillin-resistant Staphylococcus aureus Isolated from three population groups in Medellín

Introducción: Staphylococcus aureus resistente a la meticilina (SARM) es responsable de infecciones intrahospitalarias, las que constituyen una importante causa de morbilidad y mortalidad en nuestro medio, por lo cual la rápida identificación y tipificación molecular de la resistencia como el complejo SSCmec es esencial para entender la epidemiología de la infección. Objetivo: Caracterizar fenotípicamente la resistencia a meticilina y genotípicamente el casete cromosomal SSCmec en cepas de S. aureus aislados de individuos de la ciudad de Medellín mediante PCR múltiple. Materiales y métodos: A 41 aislamientos (hospitalarios y de la comunidad) de S. aureus se les estableció la resistencia a cefoxitin mediante la técnica de Kirby-Bauer y la concentración inhibitoria mínima para oxacilina. Mediante PCR convencional se les confirmó la presencia del gen mecA. Para la tipificación del complejo SSCmec se utilizó PCR múltiple para amplificar 6 loci diferentes de este gen. Resultados: A todos los aislamientos se les confirmó resistencia a meticilina y la presencia del gen mecA, de los cuales 17 fueron clasificados como SSC mec I, 1 como SSC mec II, 21 como SSC mecIV; dos aislamientos no fue posible clasificarlos. Conclusiones: Con el uso de esta técnica clasificamos el 95% de los aislamientos del estudio, encontrando una mayor prevalencia de los SSCmec I y IV. La implementación de esta técnica permite una fácil caracterización de los aislamientos SARM y un apropiado manejo de la información de los integrantes de los comités de infecciones hospitalarios, lo cual podría impactar positivamente en el tratamiento a los pacientes y el control de enfermedades infecciosas intrahospitalarias.

Introduction: Methicillin-resistant Staphylococcus aureus (MRSA) is involved in nosocomial infections, representing an important cause of morbidity and mortality. The rapid identification and molecular classification of resistance, such as the SSCmec complex, is essential to understanding the epidemiology of infection. Objective: To phenotypically characterize methicillin resistance and to genotype the SSCmec complex in S. aureus isolates collected from a cohort of patients from Medellín, Colombia. Materials and Methods: Cefoxtin resistance was evaluated in 41 S. aureus isolates, using the Kirby-Bauer method and determining the minimal bactericidal concentration of oxacillin. To confirm the presence of the mecA gene, conventional PCR was performed. The classification of the SSCmec complex was carried out by multiple PCR, amplifying 6 different loci in this gene. Results: Methicillin resistance and the presence of the mecA gene were confirmed in all isolates. A total of 17 were classified as SSCmec I, one as SSCmec II, and 21 SSCmec IV (only two isolates were not classified). Conclusions: Using this method, it was possible to classify 95% of the studied isolates, with a higher prevalence of SSCmec I and IV. The implementation of this technique allows the characterization of MRSA isolates and an appropriate management of the information by the members of the Hospital Infection Committee. Altogether, this method may have a positive impact on the treatment of patients with MRSA infections.

Rapid detection and typing of Dengue virus by single-tube nested multiplex-PCR / 第二军医大学学报

Objective: To establish a single-tube nested multiplex-PCR assay for rapid detection and typing of Dengue viruses for multiple infections with different serotypes. Methods: A pair of outer universal primers designed for all the four Dengue virus serotypes were used to amplify the mixed RNA of 1-4 dengue viral serotypes by one-step RT-PCR, and the products were used as template for nested multiplex PCR using four pairs of serotype-specific primers in the same reaction tube. The sensitivity and specificity of single-tube nested multiplex PCR assay amplifying from the mixed 1-4 serotype dengue viral RNA were subsequently compared with those amplifying from the single serotypes dengue viral RNA. Results: By optimizing the reaction condition, four specific fragments (482,119,290,and 389 bp) were successfully amplified from the mixed RNA of 1-4 serotypes dengue viruses in single tube by single-tube nested multiple-PCR. Its sensitivity and specificity amplifying from the mixed RNA of 1-4 serotypes dengue viruses were similar to those amplifying from the single serotype dengue viral RNA. The detection limit of nested multiple-PCR was 66. 068 copies/μl. Conclusion: Single-tube nested multiple-PCR method is simple, rapid, sensitive, and specific for detecting and typing dengue viruses, and it is valuable for detecting and typing of the clinical multiple infections.