Identificación del alelo HLA B*57: 01 en una población militar, de Lima-Perú / Identification of the allele HLA B*57: 01 in a military population, from Lima-Peru

El alelo HLA B*57:01 es un marcador genético asociado con la hipersensibilidad al fármaco anti-retroviral abacavir (ABC) y su frecuencia en la población peruana todavía es desconocida. El objetivo fue identificar el alelo HLA B*57:01 en una población militar de Lima, Perú. Se reclutaron 43 personas viviendo con VIH (PVV) quienes aceptaron participar a través de un consentimiento informado. La detección del alelo HLA B*57:01 se realizó mediante RPC en tiempo real (RT-PCR). Asimismo, se determinó la carga viral (CV), el recuento de linfocitos CD4 y la genotipificación del VIH. Se identificaron dos casos positivos al alelo HLA B*57:01 (4,7%). Además, uno de ellos presentó múltiples mutaciones de resistencia a los anti-retrovirales (ARV), incluyendo ABC. Se demostró por primera vez en el Perú la presencia del alelo HLA B*57:01.

The HLA B*57:01 allele is a genetic marker associated with hypersensitivity to the antiretroviral Abacavir (ABC) and its frequency in the Peruvian population is still unknown. The objective was to identify the HLA B*57:01 allele in a military population from Lima, Peru. Forty three people living with HIV (PLWH) were recruited, who agreed to participate through informed consent. Detection of the HLA B*57:01 allele was performed by real-time PCR (RT-PCR). Likewise, viral load (VL), CD4 lymphocyte count and HIV genotyping were determined. Two cases positive for the HLA B*57:01 allele (4.7%) were identified. In addition, one of them had multiple resistance mutations to antiretrovirals (ARVs), including ABC. The presence of the HLA B*57:01 allele was demonstrated for the first time in Peru.

Distribución de genotipos de virus papiloma humano de alto riesgo en mujeres y hombres atendidos en una red asistencial privada en la Región Metropolitana, Chile / Distribution of high-risk human papillomavirus genotypes in women and men attended in a private healthcare network in the Metropolitan Region, Chile

Introducción: La infección persistente por genotipos de virus papiloma humano de alto riesgo (VPH-AR) es la principal causa del cáncer cérvico-uterino en todo el mundo. Los genotipos 16 y 18 están asociados a la progresión hacia el cáncer de cuello uterino; sin embargo, otros genotipos también presentan alto riesgo oncogénico. Existe escasa evidencia respecto a la distribución de genotipos VPH-AR en la población nacional, siendo un tema que debiese ser abordado en el contexto de un creciente aumento de la inmigración e implementación del programa de inmunización en Chile desde 2015. Objetivo: Dar a conocer la distribución de genotipos de VPH-AR detectados en pacientes de ambos sexos, atendidos en la red de atención privada de Clínica Dávila de Santiago, entre los años 2014 y 2021. Metodología: Se estudiaron muestras genitales y anales provenientes de 3.642 pacientes, incluyendo ambos sexos. La genotipificación fue realizada mediante reacción de la polimerasa en cadena (RPC) en tiempo real (HPV AnyplexTM II HPV28 detection, Seegene, Korea. Resultados: La distribución global de genotipos en mujeres (porcentaje) fue: 16 (14,34%) - 31 (6,20%) - 39 (5,94%) - 58 (5,94%) - 51 (5,68%) - 53 (5,64%) - 52 (5,30%) - 56 (5,27%) - 68 (5,19%) - 66 (4,97% - 18 (3,36%) - 59 (3,29%) - 73 (2,80%) - 35 (2,54%) - 45 (2,13%) - 33 (1,53%) - 82 (1,38%) - 26 (0,49%) y 69 (0,41%). En hombres fue: 16 (8,52%) - 58 (4,39%) - 51 (8,44%) - 26 (0,42%) - 18 (3,21%) - 52 (4,47%) - 39 (5,40%) - 53 (4,56%) - 33 (1,69%) - 35 (2,03%), 73 (2,19%) - 69 (0,59%) - 45 (2,11%) - 59 (4,22%) - 68 (3,04%) - 66 (5,06%) - 31 (4,64%) - 56 (4,81%) y 82 (1,10%). Conclusiones: La distribución de los genotipos de VPH fue concordante con estudios previos nacionales. Se observó una tendencia a la reducción del genotipo 16 en el tiempo, lo cual podría relacionarse a la vacunación, implementada en los últimos años en Chile. Destaca que otros genotipos de VPH-AR tuvieron una alta frecuencia en la población estudiada por lo que sería recomendable evaluar la pesquisa ampliada de genotipos de VPH-AR para valorar el riesgo oncogénico, con fines diagnósticos y terapéuticos.

Background: Persistent infection by high-risk human papillomavirus (HR-HPV) genotypes is the main cause of cervical cancer worldwide. Genotypes 16 and 18 are associated with progression to cervical cancer, however other genotypes also present high oncogenic risk. There is little evidence regarding the distribution of HR-HPV genotypes in the national population, being an issue that should be addressed in the context of a growing increase in immigration and implementation of the immunization program in Chile since 2015. Aim: To show the distribution of HR-HPV genotypes detected in women and men, attended at the private care network of Clinica Davila, Santiago City, between 2014 and 2021. Methods: Genital and anal samples from 3,642 patients were studied, including both sexes. Genotyping was performed by real-time polymerase chain reaction (PCR) (HPV AnyplexTM II HPV28 detection, Seegene, Korea). Results: The global distribution of genotypes in women (percentage) was: 16 (14.34%) - 31 (6.20%) - 39 (5.94%) - 58 (5.94%) - 51 (5.68%) - 53 (5.64%) - 52 (5.30%) - 56 (5.27%) - 68 (5.19%) - 66 (4.97%) - 18 (3.36%) - 59 (3.29%) - 73 (2.80%) - 35 (2.54%) - 45 (2.13%) - 33 (1.53%) - 82 (1.38%) - 26 (0.49%) and 69 (0.41%). In men was: 16 (8.52%) - 58 (4.39%) - 51 (8.44%) - 26 (0.42%) - 18 (3.21%) - 52 (4.47%) - 39 (5.40%) - 53 (4.56%), 33 (1.69%) - 35 (2.03%) - 73 (2.19%) - 69 (0.59%) - 45 (2.11%) - 59 (4.22%) - 68 (3.04%) - 66 (5.06%) - 31 (4.64%) - 56 (4.81%) and 82 (1.10%). Conclusions: The distribution of HPV genotypes was consistent with previous national studies. A tendency to reduce genotype 16 over the years was observed, which could be related to the vaccination, implemented in recent years in Chile. It is remarkable that other HR-HPV genotypes had a high frequency in the population studied, so it would be advisable to evaluate an expanded screening for HR-HPV genotypes to assess the oncogenic risk, for diagnostic and therapeutic purposes.

Molecular biology analysis of 2 rare RhD variant individuals with RHD*DEL37 / 北京大学学报(医学版)

The Rh blood grouping system is a critical standardized test in transfusion medicine,espe-cially for the cases related to haemolytic transfusion reactions and neonatal haemolytic disease caused by clinical RhD blood group incompatibility.In the present case report,we presented two cases with the un-common RHD gene variation RHD*DEL37.The blood samples of the two subjects were mistakenly iden-tified as RhD-negative through conventional serological testing.Firstly,both blood samples were tested negative for the RhD antigen using traditional tube test and gel microcolumn methods.The phenotyping of RhCE were identified as ccEe and ccee for each sample,respectively.Secondly,genetic analysis was performed using polymerase chain reaction-sequence specific prime(PCR-SSP)which revealed that nei-ther sample belonging to the several common RHD gene variants which was found in Asia.Moreover,they turned out to be positive for the RHD haplotype,which indicated that exons 1-10 on one of the RHD al-leles were entirely absent.In addition,a T＞C mutation was observed at bases 1154-31 in intron 8 of the other allele,which was located at the intron 8 breakpoint.This result was obtained after further Sanger sequencing of exons 1-10 of the RHD gene.The mutant allele was designated as RHD*DEL37 by the International Society of Blood Transfusion(ISBT)and was identified as D-elute(Del)by phenotype ana-lysis.Both samples were genotyped as RHD*DEL37 and showed positive results.In summary,the true genotype of the two blood samples,of which the screening results only using serological testing method was negative,were RHD*DEL37/RHD-(RHD*01N.01).Notably,this kind of genotype was reported for the first time in Chinese population.Moreover,the two individuals did not have ties of consanguinity,indicating that some of the Chinese individuals could be carriers of the genetic mutation.Therefore,it might be necessary to further confirm the frequency of this mutation in the Chinese population and the possibility of homozygosity for this mutation.This report identifies infrequent RHD gene mutation samples by coupling molecular biology and serological methods to prevent misclassification of blood groups.Com-bining serological and molecular biology test results to determine blood group is critical in protecting pa-tients during clinical transfusion procedures.

Correlation of the emm genotyping and virulence genes with the isolation sites of Group A Streptococcus strains from children with impetigo / 中华实用儿科临床杂志

Objective:To investigate the correlation of the emm genotypes and virulence genes with the isolation sites of Group A Streptococcus (GAS). Methods:It was a retrospective study.The specimens were collected from children with impetigo in Beijing Children′s Hospital, Capital Medical University from 2006 to 2008 for GAS isolation and identification.A total of 24 GAS strains were isolated from 16 children with impetigo, among which 7 pairs of strains were isolated from the throat and skin of 7 children, and 1 pair of strains was isolated from the vulva and skin of one child, and the remaining 8 GAS strains were isolated from the skin pus samples of 8 children.Polymerase chain reaction was applied to detect the emm genotypes and 13 virulence genes ( speA, speB, speC, speF, speG, speH, speI, speJ, speK, speL, speM, smeZ and ssa). The correlation of the emm genotypes and virulence genes with the isolation sites of GAS strains was analyzed. Results:In this study, four emm genotypes were detected, including emm1.0 (15/24), emm12.0 (4/24), emm22.0 (2/24) and emm160.0 (1/24), and one subtype emm12.19 (2/24) was detected as well.The carrying rates of 13 virulence genes speA, speB, speC, speF, speG, speH, speI, speJ, speK, speL, speM, smeZ and ssa were 58.3%, 100%, 91.7%, 100%, 50.0%, 12.5%, 54.2%, 66.7%, 16.7%, 25.0%, 12.5%, 100% and 91.7%, respectively.All strains carried 5 to 11 virulence genes and they all carried speB, speF and smeZ.There were significant differences in the carrying rate of speA and speJ among the strains with different emm genotypes (all P<0.05). There was no significant difference in the distribution of virulence genes between skin isolates and pharyngeal isolates, including the 5 pairs of strains carrying the emm1.0 genotype (all P>0.05). Conclusions:The distribution of virulence gene of GAS in children with impetigo is significantly correlated with the emm genotype, rather than the isolation site.

CRISPR genotyping and regional distribution characteristics of Yersinia pestis in natural plague focus of Tibet Autonomous Region / 中华地方病学杂志

Objective:To study the clustered regularly interspaced short palindromic repeats (CRISPR) genotype of Yersinia pestis and its regional distribution characteristics in natural plague focus of Tibet Autonomous Region. Methods:A total of 125 representative Yersinia pestis strains isolated from natural plague focus in Tibet Autonomous Region at different times, regions, hosts and vectors were selected as experimental strains, and the phenol chloroform mixed extraction method was used to extract Yersinia pestis DNA. Three pairs of CRISPR primers (for YPa, YPb, YPc locus) were used to amplify the DNA of the experimental strains, and the CRISPR genotype of Yersinia pestis was determined by sequencing. Results:All 125 strains of Yersinia pestis had three CRISPR locus: YPa, YPb, and YPc. A total of 18 spacer were found, including 8 in YPa loci, 6 in YPb loci, and 4 in YPc loci. Two new types of spacers had been discovered, namely b52 and c14. CRISPR typing revealed 10 genotypes, including G1, G7, G7-b4''', G7-b52, G7-c2 -, G8, G22, G22-a4 -, G22-b4''', and G22-c14, of which 6 were newly discovered genotypes. Among the 125 experimental strains, G7 was the main genotype, accounting for 65.6% (82/125), which was distributed in 6 prefecture level citys and 1 region of Tibet Autonomous Region. Next were G22 and G7-c2 - genetypes, accounting for 14.4% (18/125) and 11.2% (14/125), respectively. G22 gene type was distributed in Nagqu, Changdu, Lhasa citys, and Ngari Prefecture, while G7-c2 - genetype was distributed in Shigatse and Shannan cities. Conclusion:The CRISPR locus of Yersinia pestis in natural plague focus of Tibet Autonomous Region is highly polymorphic, and the Yersinia pestis strains with different genotypes have obvious regional distribution characteristics.

Construction of a droplet digital PCR detection system for platelet HPA-3 and HPA-15 genotyping / 中国输血杂志

【Objective】 To establish a highly sensitive detection method of platelet HPA-3 and HPA-15 genotyping by droplet digital PCR (ddPCR), and to explore the feasibility of applying it to the detection of human platelet antigen (HPA) compatibility in maternal peripheral blood fetal free DNA. 【Methods】 For SNP mutation sites of HPA-3 and HPA-15, specific primers and MGB probes were designed, and amplification conditions such as annealing temperature and primer concentration of ddPCR were optimized to establish the optimal reaction system and clarify the test procedures. The methodological performance of the assay was evaluated, including specificity, sensitivity, repeatability and stability. ddPCR was used to detect 67 clinical blood samples, and the allele typing results were compared with the gene sequencing results. The fetal free DNA HPA antigen of 52 maternal peripheral blood samples was detected. 【Results】 The ddPCR method for detecting platelet HPA-3 and HPA-15 showed good specificity of primers and probes. The optimal annealing temperatures for HPA-3 and HPA-15 were 61.6℃ and 60.2℃, respectively. The optimal concentrations of primers were 900 nM and 700 nM respectively. The final concentration of the probe was 250 nM. The quantitative detection range of copy number was 2 to 20 000 copies, with lower limit of detection of 0.1 copies/μL, and the linearity is good. In low copy number samples, the intra - and inter batch coefficient of variation (CV) of actual detection values for HPA-3 and HPA-15 were both lower than 5%. The detection results of HPA-3 and HPA-15 genotypes of 67 blood samples were consistent with the gene sequencing results, and its application in fetomaternal platelet HPA-3, HPA-15 genotype detection met expectations. 【Conclusion】 The HPA-3 and HPA-15 ddPCR detection system constructed in this study has high accuracy, good repeatability, stability and sensitivity, and can be applied to the establishment of platelet HPA-3 and HPA-15 genotype donor pool, gene matching and fetomaternal platelet compatibility detection.

Association study between mammalian sterile 20-like kinase 1 SNPs and colorectal cancer / 安徽医科大学学报

Objective @#To explore the association between mammalian sterile 20-like kinase 1(MST1)gene poly- morphism and haplotype and the risk of colorectal cancer,rectal cancer,and colon cancer in the Han population in Baotou area by case-control association study@*Methods @#A total of 390 patients with colorectal cancer diagnosed by pathology and 413 normal physical examination population were collected,and 2 ml of peripheral blood was taken for subsequent gene genotyping.Single nucleotide polymorphisms(SNPs)of MSTI gene were screened according to the genetic polymorphism data of Chinese Han population provided by the National Center for Biotechnology Information-Haplotype Mapping database.Gene genotyping was performed by Taqman method.Logistic regression was used to calculate the association between each SNP and the risk of colorectal cancer,colon cancer,and rectal cancer under codominant,dominant,overdominant,and recessive genetic models.@*Results @#Four SNPs of MSTI gene were screened,namely rs8000,rs2234197,rs2267853,and rs6073629.Among them,SNP rs2234197 was associated with the risk of rectal cancer.Compared with the GG+AA genotype,the AG genotype could reduce the risk of rectal cancer, OR[95%confidence interval(CI)]=0.657(0.442-0.976).SNP rs8000 was associated with the risk of colon cancer.Compared with the TT+GT genotype,theGG genotype could reduce the risk of colon cancer [OR(95%CI)=0.425(0.182, -0.992)].@*Conclusion @#MSTI gene SNP rs2234197 AG genotype and SNP rs8000 GG genotype may be protective factors for rectal cancer and colon cancer,respectively.

Establishment and genotype identification of hepatic stellate cell-specific Grk2 gene knockout mouse model / 中国药理学通报

Aim To establish a stable hepatic stellate cell ( HSC ) -specific G protein-coupled receptor kinase 2 ( GRK2 ) knockout mice and provide the important animal model for further studying the biological function of GRK2 in HSC. Methods The loxP-labeled Grk2 gene mouse (Grk2

Expert consensus on the resolution level of the methods for HLA genotyping / 中国输血杂志

Human leukocyte antigen (HLA) genotyping has been widely used in establishing the database of the donors for hematopoietic stem cell, HLA matching selection between the donor and recipient, establishing the database of platelet donors with known HLA genotype, diagnosis of HLA association with diseases, genetics study and other scientific research. With the increasing number of HLA alleles and the development of HLA genotyping technology, there are some controversy in the definition of resolution for HLA genotyping, the description of the results for HLA genotyping between donor and recipient and the determination of HLA matching level. In order to improve the normalization of the definition and interpretation of HLA genotyping resolution, this expert consensus is formulated by many experts from the fields of HLA genotyping in the laboratory and clinical transplantation according to the relevant domestic and foreign literature and clinical practice. The definition of the resolution of HLA genotyping, the methods of HLA genotyping, the description of the results between donor and recipient and HLA matching determination are summarized, which will further standardize HLA genotyping technology and ensure the accuracy of the results for HLA genotyping.

Detection and genotyping of enteroviruses in 3 960 influenza-negativeinfluenza-like illness cases in Chongqing / 公共卫生与预防医学

Objective To understand the epidemiological characteristics and genotype distribution of enterovirus (EV) in influenza-negative influenza-like illness (ILI) cases in Chongqing, and to provide a scientific basis for EV prevention and control. Methods Throat swab samples of influenza-negative ILI cases were collected from surveillance sites. The samples were detected for EV using real-time RT-PCR. The VP4 regions of positive samples were amplified and sequenced for genotyping. Results A total of 3 960 influenza-negative ILI samples were collected from January to December 2021, and 316 (7.98%) of them were EV-positive. EV could be detected in influenza-negative ILI cases in Chongqing all year round. The months with high EV-positive rates were January (11.60%), April (10.56%), May (11.79%), June (12.62%), and July (10.33%). There was a statistically significant difference in the detection rate of EV in ILI cases in different regions, gender, and age groups (χ2=29.647，χ2=4.192，χ2=69.176，P<0.05). A total of 213 EV-positive cases were successfully genotyped, including 17 genotypes of EV-A, EV-B, and EV-C and 5 genotypes of HRV-B. The dominant genotypes were CV-A4 (32.86%), CV-A2 (23.00%), CA-6 (12.21%), and CA-10 (11.74%). EV-D and novel EV were not identified in this study. Conclusion EV is an important pathogen in ILI cases in Chongqing. The prevalence of EV in ILI cases in Chongqing has typical regional, seasonal and population characteristics. Prevention and control should be carried out in Chongqing according to the epidemic characteristics of EV.

Estandarización de PCR en tiempo real para detección y cuantificación de los oncogenes E7 de seis Virus del Papiloma Humano de Alto Riesgo / Real-time PCR standardization for detection and quantification of E7 oncogenes from six High-Risk Human Papillomaviruses

El cáncer de cuello uterino es causado por la infección persistente del epitelio cervical con los genotipos de alto riesgo del Virus del Papiloma Humano. Para su detección se realizan pruebas moleculares que detectan el gen L1 del VPH. Este gen puede perderse hasta en el 11 % de los casos durante la integración del ADN viral en el genoma del hospedero originando falsos negativos. Por otra parte, el oncogén E7 se expresa durante todas las etapas de progresión de la enfermedad. El objetivo de este trabajo fue estandarizar una PCR en tiempo real del oncogén E7 (E7-qPCR) para genotipificación y cuantificación de 6 VPH-AR. Los resultados muestran que la E7- qPCR amplificó VPH-16, -18, -31, -33, -35 y -45 con una alta sensibilidad con límites de detección desde 102 copias, eficiencias entre 90 y 110 %, valores R2 > 0,97 y análisis de curva de fusión que revelan productos específicos.

Cervical cancer is caused by persistent infection of the cervical epithelium with the high-risk genotypes of the Human Papilloma Virus (HR-HPV). For its detection, molecular tests are carried out that detect the L1 gene of HPV. This gene can be lost in up to 11 % of cases during the integration of viral DNA into the host genome, causing false negatives. On the other hand, the E7 oncogene is expressed during all stages of disease progression. The aim of this work was to standardize a real-time PCR of the E7 oncogene (E7-qPCR) for genotyping and quantification of 6 HR-HPV. The results show that the E7-qPCR amplified HPV-16, -18, -31, -33, -35 and -45 with high sensitivity with detection limits from 102 copies, efficiencies between 90 and 110 %, R2 values >0,97 and melting curve analysis revealing specific products.

Use of dry blots for serotyping and genotyping of dengue viruses: A pilot study

Background & objectives: Dengue (DEN) is a result of infection by one or multiple types of four dengue viruses known as Dengue virus (DENV) 1-4. Identifying circulating serotype and genotype is epidemiologically important, however, it is challenging in resource limited areas. Moreover, transporting samples from the collation site to the laboratory in appropriate condition is an exigent task. To overcome this, we evaluated the usefulness of dry blots of serum for DENV diagnosis, serotyping and genotyping. Methods: Serum samples received for diagnosis were divided into parts; one was used for providing the diagnosis. Remaining sample was distributed in three parts (100 µl each), one part was used for molecular testing and two parts were mixed with RNAlater reagent® in equal volumes and was blotted on Whatman filter paper no 3. The blots were dried and stored at 4°C and 28°C and tested for presence of dengue RNA, serotypes and genotypes after 7 days of incubation. Results: The diagnosis and serotyping results of serum sample and dry serum blots were in concordance. Out of 20 positive samples, 13 (65%) gave satisfactory sequencing results. Genotype III of DENV-1, Genotype IV of DENV 2 and Genotype I of DENV-4 were detected. Interpretation & conclusion: The results demonstrate that serum mixed with RNA protective solution and blotted on Whatman filter paper no 3 can be effectively used for diagnosis, serotyping and genotyping of DENVs. This will help in easy transportation, diagnosis and effective data generation in resource limited settings.

Genotyping analysis of human papillomavirus infection in male outpatient patients / 中国现代医生

Objective To study the human papillomavirus(HPV)infection and genotype distribution characteristics in male outpatients,and compare with female infection status,in order to provide scientific basis for the clinical development of prevention and treatment measures for male HPV infection related diseases.Methods Polymerase chain reaction(PCR)amplification followed by directed hybridization was used to detect 37 HPV genotypes in 258 male outpatients of Yangzhou Maternal and Child Health Hospital from July 2018 to December 2022.The detection results were further compared with the detection results of 1436 female physical examinees and 931 cervical exfoliated cell samples of gynecological patients suspected of HPV infection at the same time.Results There were 103 of the 258 male outpatients were positive,with an infection rate of 39.92%.Among the 103 positive samples,high-risk,low-risk,and mixed high-risk HPV infections accounted for 58.25%,20.39%,and 21.36%,respectively.Among them,59 were infected with single infection,accounting for 57.28%,44 were infected with more than double infection(multiple infection),accounting for 42.72%,and the most one had ten types infections.Single infection was mainly high-risk type,while multiple infection was mainly high-risk type and mixed high-risk type.There was no statistically significant difference in HPV infection rate,infection type,and infection status between male patients and female patients(P=0.456,0.192,0.102),but there was a statistically significant difference compared with female physical examinees(P<0.001,0.032,<0.001).The peak age of HPV infection is 20-39 years old,accounting for 74.75%,and there was no statistically significant difference in HPV detection rate among different age groups(P=0.297).33 HPV genotypes were detected in both male and female groups.The top five subtypes were HPV52,58,51,54 and 61 in male patients,while HPV57,67,69 and 83 were not detected.The top five subtypes were HPV52,16,58,53 and 61 in female groups,while HPV57,69,72 and 26 were not detected.Physical examination is the main reason of 103 male infected patients seeking medical treatment.Conclusion The HPV infection status in male is similar to that of female patients,with high-risk infection being the main type and single subtype infection being the main infection.The peak age of male patients with HPV infection is 20-39 years old.HPV52,58,51,54,61 are the most common types,and most of them are asymptomatic.Therefore,men are high-risk groups of HPV infection.It is necessary to carry out HPV detection for male outpatients.

Construction of phosphodiesterase 4D homozygous knockout mice / 安徽医科大学学报

Objective @#The CRISPR / Cas9 technology was applied to construct PDE4D homozygous knockout mice to provide a basis for in-depth investigation of PDE4D gene function and mechanism of action．@*Methods@#A vector was constructed for PDE4D gene exon 4，5 microinjected into fertilized eggs of C57BL /6J mice，and PDE4D －/ - mice were obtained after maternal breeding and offspring mating，and the mice genotypes were determined by PCR product sequencing and genotype identification techniques．Changes in morphology and function of the major organs of the mice were detected using an ultrasound imaging system and H＆E staining，and the expression of PDE4D protein in the mice was verified by Western blot assay. @*Results @#The PDE4D －/ － mouse genotype was stably inherited， the mice were small，and there were no obvious morphological and histological changes in the major organs in vivo. The PDE4D expression was reduced or largely absent in the major tissues of PDE4D heterozygous or pure knockout mice，and the knockout effect was better．@*Conclusion @#PDE4D －/ － mice were successfully established using CRISPR / Cas9 technology，and no significant physiological abnormalities were found，which could be used for disease pathogenesis and drug research using PDE4D as the target.

Clustered regularly interspaced short palindromic repeats genotyping of Yersinia pestis in Yushu Tibetan Autonomous Prefecture, Qinghai Province / 中华地方病学杂志

Objective:To learn about the clustered regularly interspaced short palindromic repeats (CRISPR) genotyping of Yersinia pestis in Yushu Tibetan Autonomous Prefecture (Yushu for short), Qinghai Province, and to explore its genetic characteristics. Methods:In this study, 44 representative strains isolated from local natural plague focus in Yushu from 1963 to 2007 were selected as experimental objects to extract DNA. Primers targeting the three CRISPR loci (YPa, YPb, and YPc) were designed for PCR amplification. The amplified products were sequenced and analyzed to identify the CRISPR spacer, and to determine the CRISPR genotypes and clusters.Results:Twenty-three spacers including 14 of YPa, 6 of YPb and 3 of YPc were observed among 44 strains, of which 2 spacers (a106 and a107) were firstly identified. According to the spacer arrays, the strains were divided into 15 CRISPR genotypes and classified into 6 CRISPR clusters which were Cb4, Cc3', Ca7, Ca7', CaΔ5' and Ca35', respectively. Among them, Ca7 was the most epidemic dominant cluster (34 strains) in Yushu.Conclusion:The CRISPR loci of Yersinia pestis in Yushu have multiple genotypes, high genetic polymorphism, and complex population structure.

Application of MassARRAY genotyping assay in the diagnosis of neonatal genetic metabolic diseases / 中华检验医学杂志

Objective:To investigate the accuracy, effectiveness and feasibility of MassARRAY genotyping assay in the diagnoses of neonatal genetic metabolic diseases.Methods:This is a retrospective study. From December 2016 to January 2020, newborns were screened by tandem mass spectrometry at the Zhejiang Newborn Screening Center, among which the data of 7 922 suspected positive cases of genetic metabolic diseases were collected. These patients were then tested for the common variants of 27 genetic metabolic diseases by MassARRAY genotyping assay, along with further testing using Sanger or next-generation sequencing used to verify and/or further search for potential variants.Results:A total of 1 408 cases were tested with MassARRAY. Among these, 307 cases were confirmed with certain genetic metabolic diseases. The detection rate of hyperphenylalaninemia was the highest, followed by primary carnitine deficiency, short acyl-coA dehydrogenase deficiency and methylmalonic acidemia. With these cases, the consistency of Sanger sequencing and MassARRAY was 100% (307/307). Another 287 cases were identified as carriers by MassARRAY with a 49.1% (141/287) consistency in reference to Sanger sequencing, mainly involving SLC22A5 and MCCC1 genes. Meanwhile, 50.8% (146/287) of these cases were found to have another variant mainly involving PAH, PTS and ACADS genes. The remaining 814 cases have no variants; 158 cases out of these patients have continuously abnormal amino acids, acyl carnitines, urine organic acid and/or other biochemical indices, and were tested by next-generation sequencing, among which 38% (60/158) were detected with two variants. In this study, a total of 513 patients with genetic metabolic disease were diagnosed, and the detection rate of MassARRAY was 59.8% (307/513). Conclusions:MassARRAY genotyping assay can be used as an early molecular screening method for neonatal genetic metabolic diseases. The detection rate is particularly high in diseases with a high concentration of hotspot variants, such as hyperphenylalaninemia and primary carnitine deficiency. The future application value of MassARRAY should be further improved by continuously optimizing its ability to identify new disease genes and potential variable sites.

Etiological analysis of clinical cases of live attenuated varicella vaccine / 中国生物制品学杂志

@#Objective To analyze the etiology of clinical cases of live attenuated varicella vaccine.Methods 64 samples of varicella vesicle fluid from 49 patients clinically diagnosed as varicella cases in phase Ⅲ clinical trial of live attenuated varicella vaccine in enterprises(the test site was Henan Province) were collected,extracted for DNA,and distinguished for wild-type strains and vaccine strains by PCR and restriction fragment length polymorphism(PCR-RFLP).Genotype was analyzed using the genotyping method recommended at the international(varicella-zoster virus,VZV) nomenclature meeting2008.Results 64 vesicle fluids were all wild-type strains(Pst Ⅰ~+Sma Ⅰ~-BssH Ⅱ~-Nae Ⅰ~-),and no vaccine-related cases occurred.All 49 isolates belonged to Clade 2.Additionally,compared with Clade 2,a synonymous mutation(T→C) in SNP18 082 was detected in all 49 isolates,and a mutation(C→A) in SNP790 was detected in one isolate.Conclusion In the clinical trial of live attenuated varicella vaccine in Henan Province,all the clinical cases were caused by infection of wild-type strain which belonged to Clade 2 genetic branch.

Research on the clustered regularly interspaced short palindromic repeats genotyping of Yersinia pestis in the natural plague foci of Hainan Tibetan Autonomous Prefecture, Qinghai / 中国热带医学

@#Abstract: Objective　To investigate the clustered regularly interspaced short palindromic repeats (CRISPR) genotypes and regional distribution of Yersinia pestis strains in the natural plague foci of Hainan Tibetan Autonomous Prefecture of Qinghai Province (referred to as "Hainan prefecture") and provide a scientific basis for plague prevention and control in this area. Methods　A total of 36 representative Yersinia pestis strains, which were isolated from different host animals and insect vectors from 1954 to 2009 in Hainan Prefecture, were selected as experimental subjects. The DNAs were extracted using the traditional sodium dodecyl sulfate decomposition and phenol-chloroform method. Three pairs of CRISPR primers (YPa, Ypb, YPc) were used for PCR amplification, sequencing and analysis of the DNA of the tested strains, respectively, as a means to identify the CRISPR genotypes of Yersinia pestis in Hainan Prefecture. Results　A total of 17 spacers were observed among 36 strains of Yersinia pestis, including 9 of YPa, 5 of YPb and 3 of YPc. All strains were divided into 5 CRISPR gene clusters (Cb2, Cb4 ', Ca7, Ca7 ', Ca35 ') and 6 genotypes (G1, G9, G22, G22-A1 ', G26-A1 ', G26-A1 'A4 -). The G26-a1 ' was the main genotype, which was distributed in Gonghe, Guide and Xinghai County, and the G22 is the second type, which was distributed in Gonghe and Guide County. Conclusions　The genetic polymorphism of CRISPR loci of Yersinia pestis strains in Hainan was high, and the regional distribution characteristics of Yersinia pestis strains with different genotypes were significant.

Etiological analysis of Yersinia pestis in Dulan County, Qinghai Province / 中国热带医学

@#Abstract: Objective　To understand the epidemic characteristics of plague and the phenotypic characteristics of Yersinia pestis strains strains in Dulan County, Qinghai Province, so as to provide theoretical reference for timely adjustment of the local plague surveillance program and prevention of plague recurrence, as well as effective experimental basis for clinical treatment and prevention. Methods　The biochemical characteristics, virulence factor identification and plasmid analysis of 23 Yersinia pestis strains isolated from Dulan County, Qinghai Province from 1964 to 1994 were studied by conventional methods and molecular biology techniques. At the same time, the different region (DFR) method was applied to study the genetic typing of 23 Yersinia pestis strains isolated from Dulan County, Qinghai Province according to 23 different regions of plague genome and the designed primers based on PMT1. Results　Among the 23 Y. pestis strains isolated from Dulan County, 22 strains of Y. pestis were palaeotypic biotypes, and biochemical types were Qinghai-Tibet Plateau type, and 1 strain was incompatible with both biotypes and biochemical types in this area. And 86.96% (20/23) of Y. pestis strains had four virulence factors (F1+, Pst I+, VW+, Pgm+). All of the tested strains produced F1 and Pst I, while 95.65% (22/23) of tested strains were positive for VW, and 86.96% were Pgm positive. All the 23 strains carried three plasmids, with a relative molecular weight (Mr) of 6×106, 45×106, 52×106, and these plasmids formed a stable plasmid spectrum: 6×106, 45×106, 52 ×106. The DFR typing results showed that Yersinia pestis could be divided into two genotypes, namely G05 and G08. Nineteen Yersinia pestis strains were G08 and four strains were G05. Conclusions　The 23 strains of Yersinia pestis isolated from Dulan County were mostly of Qinghai-Tibet Plateau type, and had stable biochemical characteristics. The virulence of Yersinia pestis was strong. The results of plasmid analysis and genotyping showed that the Yersinia pestis had the etiological characteristics of Qinghai-Tibet Plateau, which was consistent with the characteristics of Marmota himalayana plague foci in Qinghai-Tibet Plateau.

A Core Genome Multilocus Sequence Typing Scheme for Proteus mirabilis / 生物医学与环境科学（英文）

OBJECTIVE@#A core genome multilocus sequence typing (cgMLST) scheme to genotype and identify potential risk clonal groups (CGs) in Proteus mirabilis.@*METHODS@#In this work, we propose a publicly available cgMLST scheme for P. mirabilis using chewBBACA. In total 72 complete P. mirabilis genomes, representing the diversity of this species, were used to set up a cgMLST scheme targeting 1,842 genes, 635 unfinished (contig, chromosome, and scaffold) genomes were used for its validation.@*RESULTS@#We identified a total of 205 CGs from 695 P. mirabilis strains with regional distribution characteristics. Of these, 159 unique CGs were distributed in 16 countries. CG20 and CG3 carried large numbers of shared and unique antibiotic resistance genes. Nine virulence genes ( papC, papD, papE, papF, papG, papH, papI, papJ, and papK) related to the P fimbrial operon that cause severe urinary tract infections were only found in CG20. These CGs require attention due to potential risks.@*CONCLUSION@#This research innovatively performs high-resolution molecular typing of P. mirabilis using whole-genome sequencing technology combined with a bioinformatics pipeline (chewBBACA). We found that the CGs of P. mirabilis showed regional distribution differences. We expect that our research will contribute to the establishment of cgMLST for P. mirabilis.