Interpretation of results obtained using real-time fluorescence PCR based on multiplex PCR fragment analysis for ABCB1 tri-allele / 四川精神卫生

ObjectiveTo resolve the problems related to the abnormal interpretations of real-time fluorescence polymerase chain reaction （PCR） results for tri-allele， to formulate the interpretation methods of real-time fluorescence PCR by referring to multiplex PCR fragment analysis， so as to obtain an accurate， simple and cheap detection method for ABCB1 tri-allele. MethodsA total of 2 794 DNA samples were collected from Xi'an Mental Health Center from March 2020 to March 2021， and 5% of which were selected as experiments. Real-time fluorescence PCR method and multiplex PCR fragment analysis method were performed respectively. According to the comparison of Ct values of PCR curves and the comparison of base peak intensity in multiplex PCR fragment analysis， comparison and analysis were conducted on the interpretation results of the two methods， and samples with different interpretation results were verified， thereafter， PCR interpretation method was formulated. ResultsA total of 139 samples were collected， of which 120 alleles and 19 tri-allele were detected. The results of allele detection by two methods were absolutely consistent. In combination with the results of multiplex PCR fragment analysis， a method for the interpretation of real-time fluorescence PCR for 19 cases of tri-allele was developed as follows： when ∣∣Ct.G－Ct.T∣－∣Ct.G－Ct.A∣∣ in amplification curve was less than 3， the interpretation results were the combination of the base pairs with small Ct values； when ∣∣Ct.G－Ct.T∣－∣Ct.G－Ct.A∣∣ was greater than or equal to 3， the interpretation results were homozygotes from the base pairs with minimum Ct values. According to the interpretation method， the results of real-time fluorescence PCR were revised， and it showed 1 case of G/G， 1 case of A/A， 4 cases of T/G， 5 cases of T/A and 8 cases of T/T， which were consistent with the results of multiplex PCR fragment analysis. ConclusionReferring to the multiplex PCR fragment analysis method， the interpretation of ABCB1 tri-allele by real-time fluorescence PCR is developed， and the two interpretation methods are in good agreement.

Fluorescence PCR Amplification Related Technology / 中国生物化学与分子生物学报

Using fluorescence PCR (FPCR) technology to amplify DNA is an important part of modern biological research. The paper traced the invention of FPCR, through its main development, respective principles, design techniques, through to practical applications, etc. The two generations of phased methods of real-time quantitative PCR (QPCR) and digital PCR (DPCR) were mainly reviewed. QPCR that contained means of dyes, hydrolysis probe and its derivatives, hybridization probe containing molecular beacon and Yin-Yang probes, etc, dye melting curve and probe melting curve was summarized. DPCR involving chip digital PCR(cdPCR) and droplet digital PCR (ddPCR) was also included. Furthermore, the main application areas and limitation of FPCR, their characteristics of different types and future development direction were described.

Analysis of genetic characteristics of BABL/c mutant curly mice by microsatellite DNA markers / 中国比较医学杂志

Objective BALB/c mutant curly mice and normal BALB/c mice were genetically detected by microsatellite DNA marker analysis to detect the differential microsatellite loci between BALB/c mutant curly mice and normal mice.Methods 38 microsatellite DNA loci were selected and their variation in the BALB/c mutant curly mice, BALB/c mutant hairless mice and normal BALB/c mice were detected by multiplex fluorescence PCR and STR scanning genotyping.Results There were 27 the same microsatellite loci between the 38 microsatellite loci in BALB/c mutant curly mice and normal mice,and there were 11 differential loci, with a mutation rate of 28.9%(11/38). There were 30 the same sites between BABL/c mutant hairless mice and normal mice,and there were 8 different loci,with a mutation rate of 21.1%(8/38). There were also 12 differential loci between BABL/c mutant curly mice and hairless mice. Conclusions BALB/c mutant curly mice have a higher mutation rate and are significantly higher than those of hairless mice,demonstrating that the mutations in curly mice and hairless mice are two completely different mutations. These results provide reliable theoretical data for the future study and development of BALB/c mutant curly mice.

Development of Fluorescence PCR for Detection of Hand Foot Mouth Viruses with MGB Probe / 现代检验医学杂志

Objective A fluorescence PCR methods was developed to detect EV71 and CoxAl6 and other enteroviruses simultaneously,which used for hand,foot and mouth disease (HFMD) viruses in the clinical rapid diagnosis.Methods Designed specific primers and probes of the enteroviruses gene which represents one of the highly conserved regions of the virus gene,and optimized the detection system of real-time quantitative RT-PCR.The positive control template and the standard curve were constructed.Researched the limit of detection,repeatability and specificity of the products,and tested 26 positive samples and 10 negative samples which collected on June 2015.Results The results showed that this experiment obtained positive recombinant plasmid,the range of the linear relation was from 8 × 102 to 8 × 108 copies/μl,and the detection result within this range was fine.The optimal concentrations of EVUN upstream and downstream primers were 0.50 μmol/L and the MGB probes were 0.30 μmol/L,RT-PCR reaction conditions as follows:42℃ 30 min,95℃ 3 min.95℃ 5 s,60℃ 35 s,45 cycle.The limit of detection reached to 800 copies/μl.The CV of the repeatability assay was no more than 5％.Specificity was good,and no cross reaction with other infectious viruses.26 clinical positive samples and 10 negative samples were detected in this experiment,detection rate of positive samples was 100 ％ (26/26) and negative samples was 100 ％ (10/10) by fluorescent quantitative RT-PCR detection method.Conclusion The experiment demonstrated that the detection method of the fluorescent quantitative PCR for hand-foot-mouth viruses could be used for the rapid diagnosis in clinical application.

A real-time fluorescence quantitative PCR method for source identification of Crocus sativus and Carthamus tinctorius / 中国中药杂志

The specific PCR primer was designed base on ITS2 sequence in GenBank, and we developed a SYBRGreen real-time fluorescence quantitative PCR system for identification of Crocus sativus and Carthamus tinctorius source. Compared with Chinese herbal medicine DNA barcode technique, this method showed characteristics of shorter time, higher specificity and sensitivity. Using this method to detect 15 samples, 4 were C. sativus, 8 were C. tinctorius, and the other 3 samples were none of them. The result was in accordance with Chinese herbal medicine DNA barcode. This study lays the foundation for identification of related Chinese medical materials.

Comparative analysis on candida infection detection methods of vaginal secretion / 重庆医学

Objective To analyze the correlation among 3 kinds of detection methods of routine microscopic examination,fluorescence PCR nucleic acid amplification and fungal color culture in the fungal detection of vaginal secretion.Methods The patients with suspected vaginitis treated in this hospital during 2014-2016 were selected.Each 500 cases of negative and positive vaginal secretion samples by microscopic examination were collected.The candida types were identified by using the fluorescence PCR nucleic acid amplification,then 100 samples with the positive results of fluorescence PCR nucleic acid amplification for detecting fungal were performed the fungal microbial culture to verify the accuracy rate of typing results.Results The Kappa value of consistency test between fluorescence PCR nucleic acid amplification and routine microscopic examination was 0.632,the consistency between them was poor.Among 500 positive samples of vaginal secretion detected by routine microscopic examination,382 cases (76.4 ％) of Candida albicans infection were detected by fluorescence PCR nucleic acid amplification,73 cases (14.6％) were Candida glabrata infection,10 cases (2.0 ％) were Candida tropicalis infection,3 cases (0.6 ％) were Candida albicans combined Candida glabrata infection and 32 cases (6.4 ％) were other fungal infection.Among 500 negative samples by conventional microscopic examination,152 positive cases were identified by fluorescence PCR nucleic acid amplification,including 130 cases of Candida albicans,16 cases of Candida glabrata and 6 cases of Candida tropicalis.There was no statistical significant difference in positive rate between the fluorescence PCR nucleic acid amplification and CHROMAgar rapid color method (x2 =0.131,P =0.936).Conclusion For the patients with clinical manifestations and negative microscopic examination results,it is recommended to conduct fluorescence PCR nucleic acid amplification rapid type identification or fungal culture identification.

Pathogenic and clinical characteristics of hospitalized children with hand-foot-and-mouth disease / 中国感染控制杂志

Objective To understand the pathogenic distribution and epidemiological trend of hand-foot-and-mouth disease (HFMD),and provide evidence for the prevention and control of HFMD.Methods Children who were diagnosed with HFMD in a hospital between January and December 2015 were investigated,real time fluorescence PCR was used to detect enterovirus universal type EV,enterovirus 71 (EV71),and Coxsackievirus A16 (CoxA16) in specimens from children with HFMD.Positive rates and distribution of various types of EV among children of different months,genders,age groups,and infection types were analyzed.Results A total of 837 throat swab specimens from HFMD children were collected in 2015,380 (45.40％) of which were EV positive specimens.Virus typing showed that 110 (28.95 ％),7 (1.84 ％),6(1.58 ％),and 257(67.63 ％) were positive specimens for EV71,CoxA16,EV71 + CoxA16,and other types of EV.HFMD had a high prevalence since April,reached a peak in May-June,and remained high incidence in July-December.Positive rates of EV in children of different months were statistically different (P＜0.05).The age of onset was mainly in children under 3 years.Positive rates of EV and constitute ratios of different types of EV in children of different age groups were all statistically different (all P＜0.05).The positive rate of EV in severe HFMD cases was higher than common cases (65.34％ vs 27.06％,P＜0.001).The proportion of severe cases in children with EV71 infection and other types of EV infection were 90.00％ and 60.70％ respectively;children with EV71 + CoxA16 double infection were all severe cases.Constitute of EV types in children with different infection types was statistically different(P＜0.001).Conclusion In 2015,EV infection in hospitalized children with HFMD in this hospital was mainly caused by other types of EV (nonEV71 and non-CoxA16),the high prevalence season,high-risk population under 3 years of age,and severe cases should be paid high attention,prevention and treatment should be performed well.

New EMA real-time fluorescence PCR method for detection of alive Listeria monocytogenes in foods / 中国人兽共患病学报

A new EMA real-time fluorescence PCR method was developed to detect alive Listeria monocytogenes in foods.The specific primers and probe were designed based on the conserved inlA gene.The pretreatment conditions including EMA of different concentrations and irradiating times were optimized.The detection limit and inhibition rate to dead bacteria of this method were confirmed by using direct plating method.The detection specificity was evaluated by using 35 L.monocytogenes strains,25 non-L.monocytogenes strains and 92 non-Listeria strains.Simulation detection experiments were performed on 15 beverage samples and 15 cooked meat samples supplemented separately with inactivated L.monocytogenes,alive L.monocytogenes and Staphylococcus aureus.Results showed that the Ct of EMA real-time fluorescence PCR for alive L.monocytogenes was Ct=38.46-3.30 × log (R2=0.999).The detection limit was 55 cfu per reaction.Inhibition rate of DNA of inactivated strains was over 99.98％.The Ct of 35 L.monocytogenes strains were between 16.21 and 29.38,while 25 non-L.monocytogenes strains and 92 non-Listeria strains had Ct ＞35.The variation coefficient of CT was less than 5％ when the experiments were repeated.Results of 30 simulation samples were consistent with that by using standard method.The test time by using newly developed EMA real-time fluorescence PCR was shortened from 3-5 days to about 10 h.The newly developed EMA real-time fluorescence PCR method for alive L.monocytogenes is rapid,convenient,specific and sensitive and could be applyed in foods inspection.

Screening for group B Streptococcal infection in late pregnant women in Xuzhou area and its drug sensitivity analysis / 国际检验医学杂志

Objective To compare the colonization rates of group B streptococcus (GBS) detected by using fluorescent PCR and bacterial culture in late pregnant women in Xuzhou area and analyze the drug resistance of GBS .Methods The fluorescence PCR assay and bacterial culture assay for GBS were both performed for the late‐pregnant women′s vaginal swabs and anal swabs samples which were collected in this area .The diagnostic efficiency were compared between the two methods .Then the drug sensitivity test were performed for stains isolated for the bacteria culture .Results Among the 484 spcimens ,53 cases were positive detected by u‐sing fluorescent PCR ,and the positive rate was 11 .0% ,the sensitivity was 100 .0% .31 cases were detected positive by using bacte‐rial culture ,and the positive rate was 6 .4% ,the sensitivity was 59 .6% .There were statistically significant differences between the positive rate and the sensitivity of the two groups(P<0 .05) .The drug sensitivity test showed that the sensitive rates were 100 .0%to penicillin ,ceftriaxone ,vancomycin .The resistance rates to erythromycin ,clindamycin ,levofloxacin were 71 .0% ,64 .5% and 58 .1% ,respectively .Conclusion The screening rate of GBS in late pregnant women is not low in Xuzhou area .PCR is a more rap‐id ,specificific and sensitive method .Routine detection of GBS should carried out by using the method of fluorescent PCR .Resistance rate of GBS to erythromycin and clindamycin were high in Xuzhou area .More attention should be paid to the rational use of antibi‐otics to prevent drug‐resistant producing in strains of GBS .

Study on the Quinolone Antibiotic Resistance and Its Relative Genes in Campylobacter Jejuni / 现代检验医学杂志

Objective To establish a real-time fluorescence PCR method to detect the drug resistance genes of pathogenic Campylobacter jejunum in human stool samples,and investigate the relationship between quinoloneantibiotic resistance and the related genes in Campylobacter jejuni .Methods According to the gyrA and gyrB gene sequences that related with the fluoroquinolone resistance in Campylobacter jejuni ,the primers of the PCR method was designed and synthesized.A rapid real-time fluorescence PCR method to detect the drug resistance genes in Campylobacter jejuni samples was established,and the optimum reaction system and conditions were screened through an optimized approach.The developed method was com-pared with the classical drug susceptibility assay.Results It was found in the compared results that,there were 8 inconsis-tent strains of Campylobacter jejuni ,2 of the 8 strains were drug sensitive but contented the drug resistance gene,while 6 strains were drug resistant but had no drug resistant gene.Conclusion The established method can be applied to detect the drug resistance relative genes of gyrA and gyrB in Campylobacter jejuni .There was some correlation between the drug re-sistance representation and its genotype,but this point requires further studies.

Detection of HIV proviral DNA by a duplex fluorescence PCR for early diagnosis of HIV infection in infants / 中华微生物学和免疫学杂志

Objective To establish a duplex fluorescence PCR for detection of HIV proviral DNA and to evaluate its application for early diagnosis of HIV infection in infants .Methods A duplex fluores-cence PCR system was set up based on TaqMan technology for detection of human ribonuclease P ( RNase P) gene and long terminal repeat ( LTR) region of HIV.A recombinant plasmid containing the targeted gene fragment , pTG19-T, was constructed by TA cloning technique and used as the template for evaluation of sen -sitivity of the assay .Blood samples from 11 healthy individuals and 98 HIV-infected patients were collected and detected to validate the assay specificity .The assay of duplex fluorescence PCR was then carried out to detect 96 infant blood samples collected from several maternal and child health hospitals in Zhejiang province from January 2011 to September 2012 for early diagnosis of HIV infection .The results were compared with those by using the Roche HIV DNA qualitative detection kit .Results The established duplex fluorescence PCR could specifically detect HIV proviral DNA with a specificity of 100%and a detection sensitivity of 100 cps per reaction .The coincidence rate between the established assay and the Roche HIV DNA qualitative de -tection kit was 100%in the detection of 96 blood samples .Conclusion The duplex fluorescence PCR as-say showed advantages of cost-effectiveness , convenience , good specificity and accuracy with high sensitivi-ty.It could be used for early diagnosis of HIV infection in infants and also as a general technical platform for the detection of HIV proviral DNA .

ApoE genotyping by real-time fluorescence PCR / 临床检验杂志

Objective To establish a new effective method by using real-time polymerase chain reaction (PCR) to detect single nucleotide polymorphism (SNP) typing for rapid identifying apolipoprotein E alleles.Methods To determine alleles of human apolipoprotein E genetic polymorphism at Cys112Arg locus was detected by PCR melting curve analysis with fluorophore SYBR Green I. In order to increase the speciality of SNP assays, high fidelity Taq polymerase was used. The reliability of SNP typing was validated by comparison with the results of direct DNA sequencing.Results Each sample was determined by double tubes, and two melting curves were analysis. As compared the Tm value of samples with the Tm of standard substance, the apoE genotype of samples was determined. The apoE genotype of 30 samples were E3/3 (27/30) and E3/4 (3/30) respectively, which was accordant with the results of PCR-RFLP and DNA sequencing.Conclusion The presented allelic assay was specific, easy to operate and applicable for discrimination of apolipoprotein E genotyping of human blood.

Development of hepatitis C virus(HCV) fluorescence PCR (F-PCR) diagnostic kit and its clinical trial comparing with immunological method / 中国免疫学杂志

Objective:Use a new F-PCR method to develop a hepatitis C virus(HCV) diagnostic kit, test the kit through clinical trial and compare it with immunological method. Methods: Fluorescence PCR(F-PCR) is a method which combines PCR and fluorescence probe hybridization together to measure DNA/RNA. Because in-tube monitoring of fluorescence signal can be done to stand for the quantitity of PCR product. Electrophoresis and UV detection are eliminated, so after-PCR cross-contamination which causes false positive can be avoided. Results:A clinical diagnostic kit for HCV with this method is developed. 512 clinical serum samples were tested with this kit, using HCV FLISA kit from Abbott Co.and HCV Fluorescence RT-PCR kit from Biotronics Co. (B-PCR) as control. The results shows :positive rate is 30.5%,sensitivity 97.3 % and specificity 98.1% . Conclusion: F-PCR is obviously superior to ELBA, and higher than B-PCR in sensitivity. The specificity of those three kits have no statistic difference. F-PCR can be used to monitor RNA of HCV in serum, and could be useful for clinical diagnose and therapy effects monitoring.