NanoHDA： A nanoparticle-assisted isothermal amplification technique for genotyping assays

Isothermal methods, such as helicase-dependent amplification （HDA）, have an advantage over polymerase chain reaction for DNA amplification owing to their ease of operation. Here, we developed a new HDA method that is nanoparticle-assisted, termed nanoHDA. This method uses gold nanoparticles （AuNPs） to improve the sensitivity and specificity of the isothermal method. In HDA, the denaturation of DNA templates is mediated by helicases, but this method is limited by the low denaturation efficiency of helicases. In this report, AuNPs with preferential affinity for single-stranded DNA （ssDNA） were utilized to improve the denaturation efficiency of helicases. The same affinity property of nanoparticles can also enhance specificity by suppressing primer-dimer formation. This nanoHDA method was employed to genotype the KRAS gene in genomic DNA samples from colorectal cancer patients, as achieved by the hybridization of nanoHDA amplicons using the NanoBioArray chip.

Screening and identification of bioactive compounds from citrus against non-structural protein 3 protease of hepatitis C virus genotype 3a by fluorescence resonance energy transfer assay and mass spectrometry

BACKGROUND Hepatitis C virus genotype 3a(HCV G3a)is highly prevalent in Pakistan.Due to the elevated cost of available Food and Drug Administration-approved drugs against HCV,medicinal natural products of potent antiviral activity should be screened for the cost-effective treatment of the disease.Furthermore,from natural products,active compounds against vital HCV proteins like non-structural protein 3(NS3)protease could be identified to prevent viral proliferation in the host.AIM To develop cost-effective HCV genotype 3a NS3 protease inhibitors from citrus fruit extracts.METHODS Full-length NS3 without co-factor non-structural protein 4A(NS4A)and codon optimized NS3 protease in fusion with NS4A were expressed in Escherichia coli.The expressed protein was purified by metal ion affinity chromatography and gel filtration.Citrus fruit extracts were screened using fluorescence resonance energy transfer(FRET)assay against the protease and polyphenols were identified as potential inhibitors using electrospray ionization-mass spectrometry(MS)/MS technique.Among different polyphenols,highly potent compounds were screened using molecular modeling approaches and consequently the most active compound was further evaluated against HCV NS4A-NS3 protease domain using FRET assay.RESULTS NS4A fused with NS3 protease domain gene was overexpressed and the purified protein yield was high in comparison to the lower yield of the full-length NS3 protein.Furthermore,in enzyme kinetic studies,NS4A fused with NS3 protease proved to be functionally active compared to full-length NS3.So it was concluded that co-factor NS4A fusion is essential for the purification of functionally active protease.FRET assay was developed and validated by the half maximal inhibitory concentration(IC50)values of commercially available inhibitors.Screening of citrus fruit extracts against the native purified fused NS4A-NS3 protease domain showed that the grapefruit mesocarp extract exhibits the highest percentage inhibition 91%of protease activity.Among the compounds identified by LCMS analysis,hesperidin showed strong binding affinity with the protease catalytic triad having S-score value of-10.98.CONCLUSION Fused NS4A-NS3 protease is functionally more active,which is effectively inhibited by hesperidin from the grapefruit mesocarp extract with an IC50 value of 23.32μmol/L.

牡丹江地区女性宫颈感染人乳头瘤病毒基因分型研究

目的明确牡丹江地区女性宫颈感染人乳头瘤病毒(Human Papillomavirus,HPV)基因型的分布特征。方法应用PCR-反向点杂交法(PCR-RDH)对牡丹江地区642例女性宫颈上皮细胞标本进行23种HPV基因型的分型检测。结果642例中共检出21种HPV基因型(HPV73、82型未检出),HPV感染阳性者226例,总感染率为35.20%;高危型HPV感染126例,阳性率为19.63%,排列前四位的高危基因型依次为HPV16、52、58、51型,低危型感染23例,阳性率为3.58%,以HPV6、43型为主,混合型感染77例,阳性率为11.99%;感染类型以单一HPV基因型感染为主,阳性率为23.20%,占65.93%,其中二重HPV感染阳性率为6.86%,占19.47%,三重及以上混合HPV感染阳性率为5.14%,占14.60%;≤20岁、21~30岁、31~40岁、41~50岁、51~60岁和>60岁6个年龄组中HPV感染阳性率分别为28.57%、40.91%、39.62%、32.14%、32.26%和23.81%。结论 HPV16、52、58、51型是牡丹江地区女性宫颈感染HPV的主要高危基因型,HPV感染率以21~30、31~40岁组中最高,分别为40.91%、39.62%。

A Rapid and Cost-Effective Method for Genotyping Genome-Edited Animals：A Heteroduplex Mobility Assay Using Microfluidic Capillary Electrophoresis

The recent emergence and application of engineered endonucleases have led to the development of genome editing tools capable of rapidly implementing various targeted genome editions in a wide range of species.Moreover,these novel tools have become easier to use and have resulted in a great increase of applications.Whilst gene knockout（KO） or knockin（KI） animal models are relatively easy to achieve,there is a bottleneck in the detection and analysis of these mutations.Although several methods exist to detect these targeted mutations,we developed a heteroduplex mobility assay on an automated microfluidic capillary electrophoresis system named HMA-CE in order to accelerate the genotyping process.The HMA-CE method uses a simple PCR amplification of genomic DNA（gDNA） followed by an automated capillary electrophoresis step which reveals a heteroduplexes（HD） signature for each mutation.This allows efficient discrimination of wild-type and genome-edited animals down to the single base pair level.

Novel CagA ELISA exhibits enhanced sensitivity of Helicobacter pylori CagA antibody

AIMTo develop a novel Helicobacter pylori (H. pylori) CagA antibody enzyme-linked immunosorbent assay (ELISA) suitable for detecting serum anti-CagA antibodies with high sensitivity.METHODSRecombinant East Asian-type CagA protein was purified and immobilized for ELISA. Serum samples from 217 Vietnamese individuals (110 H. pylori-infected and 107 uninfected individuals) were applied. Conventional ELISA from Western-type CagA and our East Asian-type CagA ELISA were evaluated by comparing 38 subjects with the Western-type genotype and 72 subjects with the East Asian-type cagA genotype. Histological scores of the gastric mucosa were determined using the updated Sydney System to examine the relationship with anti-CagA antibody titers.RESULTSRecombinant 70-100 kDa fragments were immobilized on the ELISA plate. In ROC analysis, the area under the curve of our East Asian-type CagA ELISA was comparable to that of conventional CagA ELISA. The sensitivity of the two ELISAs differed depending on the cagA genotype. The sensitivity of East Asian-type CagA ELISA was higher for subjects infected with East Asian-type cagA H. pylori (P < 0.001), and the sensitivity of the conventional CagA ELISA tended to be higher for subjects infected with Western cagA H. pylori (P = 0.056). The titer of anti-CagA antibody tended to correlate with monocyte infiltration scores (r = 0.25, P = 0.058) and was inversely correlated with H. pylori density (r = -0.26, P = 0.043).CONCLUSIONThe novel ELISA is useful to detect anti-CagA antibodies in East Asian countries, and the titer may be a marker for predicting chronic gastritis.

Investigation of Genetic Variation of B-G Gene with PCR-SSCP and RFLP in Chinese Indigenous Chickens

To reveal genetic variation of MHC B-G gene at Chinese native chickens, two PCR primer pairs were designed to hybridize specifically with conserved sequences surrounding hypervariable regions within the B-G gene and used to amplify two DNA fragments in ten Chinese indigenous chicken breeds and one introduced breed. The fragments were cloned and sequenced to assure that the expected sequences of chicken B-G gene were isolated. Of which the 189 bp fragment encompassing the most variable region within exon 2 of B-G gene was employed for PCR-SSCP assay, this method provided evidence for the presence of at least 56 B-G genotypes in the Chinese chickens sampled. It revealed a high degree of diversity in B-G genes of Chinese local breeds; particularly, high variation of B-G gene was confirmed with the presence of 48 B-G genotypes within Tibetan chicken population. Not only can the B-G genotypes be used to preliminarily screen new B-G alleles, but also they would be utilized to investigate MHC haplotypes and matched unrelated donors for bone marrow transplantation in immune researches. Another fragment of 401 bp size spanning over partial intron 1 and exon 2 of B-G gene was employed for PCR-RFLP analysis with two restriction enzymes of Msp Ⅰ and Tas Ⅰ in the breeds sampled. In this part of the gene, three novel SNPs were detected at the two restriction sites. It was more generally found the transition of two nucleotides of A294G and T295C occurred at Tas Ⅰ restriction site, and consequently led to a non-synonymous substitution of asparagine into serine at position 54 within the deduced amino acid sequence of immunoglobulin variable-region-like domain encoded by the exon 2 of B-G gene. It was observed at rare frequency that an alone mutation of A294G occurring at the site, which also caused the substitution of amino acid, asparagine 54-to-serine; and we haven＇t found only single mutation occurred at position 295 of the restriction site. At the Msp Ⅰ site, the transversion of G318C led to a non-synonymous substitution, glutamine 62-to-histidine. The variations at expression level caused by the genetic variability of B-G gene may bring about the changes in immune specificity of B-G antigen finally. Furthermore, two alleles, A and B, were identified at Msp Ⅰ and Tas Ⅰ loci of B-G gene, respectively. The allele frequencies were estimated, which gave a nonsymmetrical distribution either in the eight Chinese local breeds or in the introduced breed. By comparison, allele A at Msp Ⅰ locus was tended to be dominative, while, the allele B at Tas Ⅰ locus was tended to be prevalent in the breeds analyzed. It is concluded that the genetic variability of B-G gene revealed by the PCR-SSCP and RFLP assays in Chinese native chickens provide molecular data for further investigating the varied immune functions of B-G antigen; and the PCR-RFLPs at Msp Ⅰ and Tas Ⅰ loci of B-G gene might be used as genetic markers in selecting for the traits of disease resistance in chicken breeding.

Transfusion-transmitted virus co-infection in other types of hepatitis and its genotypes

OBJECTIVE: To identify the influence of transfusion transmitted virus (TTV) co-infection in other virus infected patients and its genotypes. METHODS: A conservative sequence of ORFl in the TTV genome was selected as primers and TTV DNA was measured in students and other hepatitis patients by using microplate nucleic acid hybridization and ELISA. The results were statistically analyzed. Nucleotide sequence of divergence >50% was used as color probe for distinguishing TTV genotypesⅠorⅡ. RESULTS: TTV DNA was detected in the sera from 2 (3.3%) of 60 students, 2 (14.3%) of 14 patients with non A-non E hepatitis, 6 (12%) of 50 patients with chronic hepatitis B, and 4 (16%) of 25 patients with liver cirrhosis, respectively. Statistical difference was observed between the patient group and the student group (P<0.05), but no significant difference in age, gender, serum ALT levels and TBiL between TTV DNA positive and negative patients (P>0.05). TTV genotype Type Ⅰ was by far the most frequent viral genotype (66.7%), followed by type Ⅱ (25%), and mixed infection (8.3%). CONCLUSIONS: These results suggest that the routes of TTV infection may be similar to those of HBV and HCV, and concurrent infection with HBV, HCV are common. TTV co-infection could not affect the clinical features of patients with liver diseases and the pathological process. TTV is not a main causative factor for patients with non A-non E hepatitis. Further study is needed to clarify the role of TTV in patients with non A-non E hepatitis.

Head-to-head comparison of 7 high-sensitive human papillomavirus nucleic acid detection technologies with the SPF10 LiPA-25 system

Background:The SPF10 LiPA-25 system for human papillomavirus(HPV)detection with high analytical perfor-mance is widely used in HPV vaccine clinical trials.To develop and evaluate more valent HPV vaccines,other comparable methods with simpler operations are needed.Methods:The performance of the LiPA-25 against that of other 7 assays,including 4 systems based on reverse hybridization(Bohui-24,Yaneng-23,Tellgen-27,and Hybribio-16)and 3 real-time polymerase chain reaction(PCR)assays(Hybribio-23,Bioperfectus-21,and Sansure-26),was evaluated in selected 1726 cervical swab and 56 biopsy samples.A total of 15 HPV genotypes(HPV 6,11,16,18,31,33,35,39,45,51,52,56,58,59,and 66)were considered for comparison for each HPV type.Results:Among the swab samples,compared to LiPA-25,compatible genotypes were observed in 94.1%of samples for Hybribio-23,92.8%for Yaneng-23,92.6%for Bioperfectus-21,92.4%for Hybribio-16,91.3%for Sansure-26,89.7%for Bohui-24,and 88.0%for Tellgen-27.The highest overall agreement of the 15 HPV genotypes combined was noted for Hybribio-23(κ=0.879,McNemar’s test:P=0.136),followed closely by Hybribio-16(κ=0.877,P<0.001),Yaneng-23(κ=0.871,P<0.001),Bioperfectus-21(κ=0.848,P<0.001),Bohui-24(κ=0.847,P<0.001),Tellgen-27(κ=0.831,P<0.001),and Sansure-26(κ=0.826,P<0.001).Additionally,these systems were also highly consistent with LiPA-25 for biopsy specimens(all,κ>0.897).Conclusions:The levels of agreement for the detection of 15 HPV types between other 7 assays and LiPA-25 were all good,and Hybribio-23 was most comparable to LiPA-25.The testing operation of HPV genotyping should also be considered for vaccine and epidemiological studies.

Genotyping for Kidd, Kell, Duffy, Scianna, and RHCE blood group antigens polymorphisms in Jiangsu Chinese Han

Background Molecular testing is more precise compared to serology and has been widely used in genotyping blood group antigens. Single nucleotide polymorphisms （SNPs） of blood group antigens can be determined by the polymerase chain reaction with sequence specific priming （PCR-SSP） assay. Commercial high-throughput platforms can be expensive and are not approved in China. The genotype frequencies of Kidd, Kell, Duffy, Scianna, and RhCE blood group antigens in Jiangsu province were unknown. The aim of this study is sought to detect the genotype frequencies of Kidd, Kell, Duffy, Scianna, and RhCE antigens in Jiangsu Chinese Hart using molecular methods with laboratory developed tests. Methods DNA was extracted from EDTA-anticoagulated blood samples of 146 voluntary blood donors collected randomly within one month. Standard serologic assay for red blood cell antigens were also performed except the Scianna blood group antigens. PCR-SSP was designed to work under one PCR program to identify the following SNPs： JK1/JK2, KEL 1/KEL2, FYA/FYB, SC1/SC2, C/c and E/e. Results Serologic antigen results were identical to the phenotypes that were predicted from genotyping results. The allele frequencies for Jk^＊01 and Jk^＊02 were 0.51 and 0.49, respectively; for Fy^＊A and Fy^＊B 0.94 and 0.06; for RHCE^＊C and RHCE^＊c 0.68 and 0.32; and for RHCE^＊E and RHCE^＊e 0.28 and 0.72. Among 146 blood donors, all were KEL^＊02/ KEL^＊02 and SC^＊01/SC^＊01, indicating allele frequencies for KEL^＊02 and SC^＊01 close to 1.00. Conclusions The use of PCR-SSP working under the same condition for testing multiple antigens at the same time is practical. This approach can be effective and cost-efficient for small-scale laboratories and in developing counties. These molecular tests can be also used for identifying rare blood types.

Diagnostic Performance of the GenoType MTBDRplus and MTBDRs/Assays to Identify Tuberculosis Drug Resistance in Eastern China

Background： The WHO recently has recommended the GenoType MTBDRph/s version 1.0 and MTBDRs/version 1.0 assays for widespread use in countries endemic with drug-resistant tuberculosis. Despite this, these assays have rarely been evaluated in China, where the burden of drug-resistant tuberculosis is among the highest globally. Methods： Mycobacterium tuberculosis clinical isolates were obtained between January 2008 and December 2008. Isolates were tested for drug resistance against rifampicin （RFP） and isoniazid （INH） using the GenoType MTBDRplus assay and drug resistance against ethambutol （EMB）, ofloxacin （OFX）, and kanamycin （KM） using the Genotype MTBDILsl assay. These results were compared with conventional drug-susceptibility testing （DST）. Results： Readable results were obtained from 235 strains by GenoType MTBDRphts assay. Compared to DST, the sensitivity of GenoType MTBDRplus assay to detect RFP, INH, and multidrug resistance was 97.7%, 69.9%, and 69.8%, respectively, whereas the specificity for detecting RFP, INH, and multidrug resistance was 66.7%, 69.2%, and 76.8%, respectively. The sensitivity and specificity of the GenoType MTBDRsl assay were 90.9% and 95.2% for OFX, 77.8% and 99.5% for KM, 63.7% and 86.4% for EMB, respectively. Mutations in codon S531L of the rpoB gene and codon S315T1 ofKatG gene were dominated in multidrug-resistant tuberculosis （MDR-TB） strains. Conclusions： In combination with DST, application of the GenoType MTBDRplus and MTBDRsl assays may be a useful supplementary tool to allow a rapid and sale diagnosis of multidrug resistance and extensively drug-resistant tuberculosis.

Programmable microfluidic genotyping of plant DNA samples for marker-assisted selection

As demands to maintain the global food production continue to mount,multinational seed companies are turning to new DNA marker technologies to accelerate the rate of plant breeding and crop improvement.The key to widespread adoption of molecular breeding is the availability of flexible and cost-effective tools that can perform combinatorial and high-throughput genotyping of single-nucleotide polymorphisms(SNPs)to guide the crop development process.Toward this end,we have developed a programmable,droplet-based microfluidic device for genotyping maize genomic DNA.A unique feature of the microfluidic platform is the nano sample processors(NSPs),which allow the device to sequentially load an unrestricted number of unique DNA samples using only two inlets,overcoming the current limitation to the number of sample inputs due to small device footprint.Direct and programmable droplet generation within the device allows each sample to be genotyped against a panel of markers on demand.Moreover,we have successfully implemented the Invader assay for SNP genotyping in flowing,50-nL droplets,thus achieving significant reduction in consumption of reagents per reaction as compared with conventional genotyping platforms.As a demonstration,we performed 240 Invader reactions(testing 8 DNA samples against 10 SNP markers)and achieved greater than 93% accuracy in SNP calling of plant DNA samples in a single droplet-based experiment.

Super-assembly of integrated gold magnetic assay with loopmediated isothermal amplification for point-of-care testing

With the increasing global threat of various diseases and infections,it is essential to develop a fast,low-cost,and easy-to-use point-of-care testing(POCT)system for inspections at all levels of medical institutions and self-examination at home.In this work,gold magnetic nanoparticles(GMNPs)are used as the key material,and a rapid visual detection method is designed through integrating loop-mediated isothermal amplification(LAMP)and lateral flow assay(LFA)biosensor for detecting a variety of analytes which includes whole blood,buccal swabs,and DNA.It is worth to note that the proposed method does not need DNA extraction.Furthermore,uracil DNA glycosylase(UDG)is employed to eliminate carrier contamination for preventing false positive results.The whole detection process can be finished within 25 min.The accuracy of detection is measured by assessing the polymorphisms of the methylenetetrahydrofolate reductase(MTHFR)C677T.The detection limit of the newly developed extraction-free detection system for MTHFR C677T is 0.16 ng/μL.A preliminary clinical study of the proposed method is carried out by analyzing 600 clinical samples(including 200 whole blood samples,100 buccal swabs,and 300 genomic DNA samples).The results indicate that the proposed method is 100%consistent with the sequencing results which provides a new choice for POCT and shows a broad application prospect in all levels of medical clinics and at home.