Newborn screening plays an important role in the 3-tiered system of prevention and control for birth defects in China.With the rapid development of newborn screening and diagnosis,quality control system including internal quality control and external quality assessment should be optimized and improved,and attention should be paid to pre-experimental quality control and management of positive patients as well.Widespread application of tandem mass spectrometry in newborn screening and diagnosis for inherited metabolic diseases greatly enhanced the detection capability and efficiency.The rapid development of molecular diagnosis techniques will confront new challenges and troubles.

The physiological and biochemical conditions of adults are relatively stable,while children are in the period of rapid growth and development with all kinds of physical and chemical indicators constantly changing,which brings great challenge to the pediatric clinical laboratories.In addition,children in different periods have different disease spectrum,among which many diseases are particular to children.This opens a new world for the pediatric laboratories.In this paper,a brief overview of problems,characteristics and future development of pediatric laboratory medicine is made.

Newborn screening of inherited metabolic diseases by tandem mass spectrometry is flourishing in our country.With expansion of the coverage and spectrum of diseases, it is important to strengthen the quality management, optimize the performance and reduce the false rate currently, such as quality assurance of the specimens, quality control of the process, quality verification of the test procedure, quality evaluation of the laboratory and quality optimization of the interpretation method. Along with development of the equipment, software and project, promotion of regional collaboration by data and experience sharing will be more critical in the future, and national neonatal screening by tandem mass spectrometry will step into a new stage.

Objective To establish the specific 16S 23S rRNA gene spacer regions map of different bacteria by PCR, RFLP(restriction fragment length polymorphism ),DNA clone and sequences analysis. Methods A pair of primer was selected from highly conserved sequences adjacent to the 16S 23S rRNA spacer region. The farget rRNA regions from 61 strains of standard bacteria and corresponding clinical isolates representing for 20 genera and 26 species were amplified by PCR,and thereafter analyzed RFLP, DNA clone and sequences analysis.Meanwhile, all the specimens were examined by bacterial culture and PCR RFLP analysis. Results 26 different standard strains presented one band,two bands,three bands and more than three bands respectively, the sensitivity of which reached 2.5 CFU and had no cross reaction to the human genomic DNA,fungus and virus.14 species could be distinguished immediately by PCR, other 10 species must be identified by further Hinf I or Alu I digestion. K.pneumoniae and E.durans differentiate only at the site of 779 th nucleotide according to the sequence analysis, and only one enzyme Xma III could discriminate them.15 specimens from 42 septicemic neonates were blood culture positive and the positive rate was 35.7%. However, 27 specimens were positive by PCR and the positive rate was 64.2%,which was significantly higher than that of the blood culture( P

Objective To investigate the relationship between dexamethasone and brain derive neurotrophic factor gene in experimental bacterial meningitis. Methods To construct the models of bacterial meningitis in 3 weeks old rats(n=33),then 23 of them were administed antibiotic and antibiotic plus dexamethasone respectively. BDNF mRNA and TrkB mRNA in brain was detected by in situ hybridization methods,respectively. Results After administration antibiotics, the expression of BDNF mRNA and TrkB mRNA in brain tissue were less than that in brain after infection 5 d(P 0.05), the expression of TrkB mRNA was stronger than that in brain at 5 d after infection(P

Objective To detect and differentiate six major human herpesviruses DNA by PCR, RFLP, DNA clone and sequence analysis. Methods Based on the sequence of well coonserved regions of the DNA polymerse gene in human herpesvimses, we synthesized two pairs of primers, including one pair designed to amplify herpes simplex virus type 1 and 2, Epstein Barr virus and cytomegalovirus, other pair of primer to varicella zoster virus and human herpesvirus 6 by PCR. Identification of the virus species was achieved through restriction enzyme digestion with BamHI and BstUI. Results The products of six human herpesviruses after PCR amplification were from 510bp to 592bp and allowed characterization of herpesvirus type with restriction endonulease analysis. The sensitivity could reach 0.1fg DNA and had no cross reaction to human genomic DNA, bacteria, fungas and virus. 89 cerebrospinal fluids(CSF) and 75 blood specimens were tested for the presence of hepersvirus DNA by this PCR assay, in which 13(14.6%) CSF and 26(34.7%) blood specimens were positive. Comparatively, 10(13.3%) were positive by ELISA for HSVⅠ/Ⅱ,EBV,CMV IgM in blood specimens and significantly lower than that of the PCR rate (P

Objective To analyze the single nucleotide polymorphisms(SNPs) in promoter region,5' untranslated region,exon 1 and haplotypes of mannose binding-lectin(MBL) gene in Han nationality and Hui etheic children in China.Methods Sixty nine Hui ethnic children from Ningxia Huizu Auto.Reg.and 105 Han children from Zhejiang Prov.were enrolled in the present study.Whole blood samples(1.5 ml) were collected into potassium-EDTA tubes.SNPs in promoter region,5' untranslated region and exon 1 of MBL gene were determined by sequence analysis using BigDye Mix 3730 genetic analyzer,and genetic analysis was performed using SHEsis software.Results The variant allele frequencies at-221 sites in Han and Hui objects were 0.091 and 0.123,respectively,with no difference between the two groups(?2=0.684,P=0.408).No mutation was found at sites +223 and +239 in exon 1 of MBL gene in the study.The variant frequency at +230 site in Hui children(0.268) was significantly higher than that in Han objects(0.167,?2=5.223,P=0.022).The most common haplotype was YA,and the frequencies of YA haplotype in Han and Hui ethnic were 0.770 and 0.669,respectively,with significant difference between the two groups(?2=4.312,P=0.038).Conclusion The variant allele frequency at +230 sites in exon 1 in Hui ethnic children is higher than that in Han subjects.The most common haplotype is YA,and the frequency of YA haplotype is higher in Han children than that in Hui subjects.

Objective To improve the speed and accuracy of bacteria detection, and develop the test of 16S rRNA genes PCR amplification plus gene chip hybridization to diagnose neonatal sepsis. Methods Bacterial 16S rRNA genes were detected in blood and CSF samples of 125 suspected neonatal sepsis, and the results were compared with blood culture, CSF culture, and non-specific diagnostic parameters (WBC, PLT, CRP). 30 non-infectious neonates were regarded as the negative control group. Gene chip test were performed by extraction of DNA, primers and probes design, PCR amplification, preparation of gene chip, hybridization, laser scan and reading of the results. 18 specific probes, including universal 1, universal 2, Gram positive bacterial probe, Gram negative bacterial probe 1, Gram negative bacterial probe 2, Staphylococcus aureus, Staphylococcus epidermidis, CoNS (Coagulase Negative Staphylococcus), Escherichia coli, Haemophilus influenzae, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Bacteroides fragilis, Bacillus, Meningococcus, Corynebacterium, Propionibacterium, were used in the test. Results The positive rate of PCR test was 51.2% in 125 blood samples, and was significantly higher than the positive blood culture (25.6%), or the indicator of two abnormal non-specific parameters (32.8%) (P

Objective To establish a method for rapid diagnosis and identification of seven species human herpesviruses infection.Methods Primers and oligonucleotide probes were designed and synthesized based on the highly conserved regions of the DNA polymerase genes in human herpesviruses, namely herpes simplex virus type 1 (HSV-1),HSV-2,varicella-zoster virus (VZV),Epstein-Barr virus (EBV),cytomegalovirus (CMV),and human herpes virus 6 (HHV-6A/6B).DNA microarrays were made by printing the oligonucleotide probes on the special glass slides.A total of 282 blood specimens from children with suspected infection were analyzed by this DNA microarray technique,and the results were compared with those of TaqMan PCR.Results The products of the seven human herpesviruses after PCR amplification could be used to identify the virus species with DNA microarrays.The detection limits were 10 copies/?l for HSV-1,HSV-2,VZV,EBV,CMV,HHV-6A,and HHV-6B,respectively.The assay did not show cross-reaction to the DNA extract of hepatitis B virus,staphylococcus aureus,E.coli,Candia albicans and human genome.Among the 282 samples,59 were positive for human herpesviruses DNA.Compared with those of TaqMan PCR,the sensitivity and specificity of the microarray assay were 96.7% and 99.5%,respectively,and the index of accurate diagnosis was 0.962.Conclusions This DNA microarray for identifying human herpesviruses species is specific and sensitive,and may serve as an efficient technique for simultaneous detection and species identification of human herpesviruses in clinical specimen.

Objective To explore a rapid diagnostic method in neonatal sepsis and bacterial me- ningitis. Methods The primers and TaqMan probes were designed and synthesized based on the sequences of bacterial 16S rRNA gene. Nineteen bacterial strains, 3 different viruses, fungus and human genomic DNA were tested by FQ-PCR assay. Blood specimens and CSF from 195 cases of suspected septicemia were detected with both TaqMan PCR assay and blood or/and CSF culture. Results The FQ-PCR showed very high sensitivity and specificity and was able to detect at least 10 copies of 16S rRNA gene equivalent to 1~2 copies bacterium. No cross-reaction was found with human genomic DNA, other fungus and viruses. Among the 195 cases, the positive rate by FQ-PCR was 12.8%(25 cases) and 7.1%(15 cases) by blood culture ( P