Development of TaqMan-based Real-time PCR Assay for Detecting Transmissible Gastroenteritis Virus and Its Application in Vaccine Evaluation

[Objective] This study aimed to establish a TaqMan-based real-time PCR assay for detecting transmissible gastroenteritis virus （TGEV）. [Method] Primers and a probe were designed according to the conserved sequence of N gene in TGEV genome. After gradient dilution, the recombinant plasmid harboring the N gene was used as a standard for real-time PCR assay to establish the standard curve. [Re- sult] The results showed that the established real-time PCR assay exhibited a good linear relationship within the range of 102-10^10 copies/ul; the correlation coefficient was above 0.99 and the amplification efficiency ranged from 90% to 110%. The de- tection limit of real-time PCR assay for TGEV was 10 copies/μl, suggesting a high sensitivity; there was no cross reaction with other porcine viruses, indicating a good specificity; coefficients of variation within and among batches were lower than 3%, suggesting a good repeatability. The established real-time PCR method could be ap- plied in quantitative analysis and evaluation of the immune efficacy of TGEV vac- cines and detection of TGEV in clinical samples. [Conclusion] The TaqMan-based real-time PCR assay established in this study is highly sensitive and specific, which can provide technical means for the epidemiological survey of TGEV, development of TGEV vaccines and investigation of the pathogenesis of TGE.

Advances in the Detection of Mycoplasma hyopneumoniae by Polymerase Chain Reaction (PCR) Technology

Mycoplasma hyopneumoniae is an important pathogen causing Mycoplasmal pneumonia of swine, which generally causes secondary infections and mixed infections, thus seriously threats the development of swine industry and resulting in huge economic losses. Using PCR technology has very important significance to the correct diagnosis of Mycoplasmal pneumonia at the early stage. In this paper, specific target genes of Mycoplasma hyopneumoniae, methods for clinical sample collection, key technical factors of DNA sample processing method, and the research progress, main advantages and disadvantages, and application of general PCR technology, multiple PCR technology, nested-PCR technology, real-time fluorescence quantitative PCR technology, gene chip detection technology and loop-mediated isothermal amplification in detection of Mycoplasma hyopneumoniae were summarized, which provided convenience for the effective diagnosis and prevention of Mycoplasmal pneumonia of swine.

Detecting Factor Ⅺ Deficiency in Holstein Cattle Using PCR Analysis

[Objective] This study established a method to detect Factor Ⅺ by polymerase chain reaction analysis.[Method]A pair of primers was designed and synthesized according to sequences of FⅪ gene in Holstein calves,published in Genbank. Polymerase chain reaction was used to analyze FⅪ deficiency of 576 Holstein calves in Henan,and the result was verified by DNA sequencing. [Result] We detect 576 cows,which include two carriers and one F Ⅺ deficiency,and the result was consistent with the DNA sequencing. The frequency of the FⅪ mutant allele was 0.3%,the carrier was 0.3%,the prevalence was 0.2%.[Conclusion]A method detecting FⅪ by polymerase chain reaction analysis was established. This method is not only simple and convenient,but also has a high accuracy and low cost,which is more suitable for large-scale FⅪ investigation.

Establishment of Multiplex PCR for Simultaneous Detection of Four Venereal Pathogens

Venereal diseases are considered to be the most prevalent infectious diseases in the worldwide. China is now faced with a year-by-year increasing incidence of sexually transmitted diseases （STD）, which are spreading from high-risk groups to the general population. Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum and herpes simplex virus-2 （HSV-2） are always regarded as the most common venereal pathogens. The ＂golden standard＂ for testing Neisseria gonorrhoeae remains to be bacteria culture or microscopic examination.

Combination of Loop-Mediated Isothermal Amplification Assay and Nested PCR for Detection of Borrelia burgdorferi sensu lato in Human Serum Samples

A set of universal loop-mediated isothermal amplification （LAMP） primers targeting the flo gene was designed to detect Borrelia burgdorferi sensu lato （B. burgdorferi s.I.） in human samples. The sensitivity of LAMP was 20 copies/reaction, and the assay did not detect false positives among 11 other related bacteria. A positive LAMP result was obtained for 9 of the 24 confirmed cases and for 12 of 94 suspected cases. The positive rate of LAMP was the same as that of nested PCR. The LAMP is a useful diagnostic method that can be developed for rapid detection of B. burgdorferi s.I. in human sera. Combination of the LAMP and nested PCR was more sensitive for detecting B. burgdorferi s.I. in human serum samples.

Novel PCR primers to diagnose visceral Leishmaniasis using peripheral blood, spleen or bone marrow aspirates

Objective: To establish a suitable method of diagnosis of visceral Leishmania sis(VL) using peripheral blood, spleen or bone marrow aspirates. Methods: Peripheral blood, bone marrow and spleen aspirate samples were collected from clinically suspected VL patients(n=26). A new PCR primer pair(MK1F/R) was designed targeting kinetoplast mini circle DNA sequences of Leishmania donovani, and Leishmania infantum, and was used to diagnose VL along with some other established primers for VL in polymerase chain reactions. Test was validated by comparing with several other diagnostic methods. Results: The designed primer set showed 100% specificity and 98% sensitivity in detecting VL using blood samples, when compared with more invasive samples: bone marrow or spleen aspirates. Conclusions: The newly designed primer MK1F/R could be a better alternative for PCR based diagnosis of VL using less invasive sample, peripheral blood instead of bone marrow or spleen aspirates.

Study on PCR rapid molecular detection technique of Meloidogyne vitis

Meloidogyne vitis is a new root-knot nematode parasitic on grape root in Yunnan Province,China.In order to establish a rapid,reliable and specific molecular detection method for M.vitis,the species-specific primers were designed with rDNA-ITS(ribosomal DNA internal transcribed spacer)gene fragment as the target.The reaction system was optimized and the reliability,specificity and sensitivity of primer were testified,therefore,a rapid PCR detection method for M.vitis was established.The result showed that the optimal annealing temperature of the primers was 53℃,which was suitable for the detection of different life stages of M.vitis.Specificity test showed that the specific fragment size of 174 bp was obtained from M.vitis,but other five non-target nematodes did not have any amplification bands,thus effectively distinguish M.vitis and the other five species,and could specifically detect the M.vitis from mixed populations.Sensitivity test showed that this PCR technique could detect the DNA of a single second-stage juvenile(J_(2))and 10^(-4)female.Futhermore,this PCR technique could be used to detect directly M.vitis from soil samples.The rapid,sensitive and specific PCR molecular detection technique could be used for the direct identification of a single J_(2)of M.vitis and the detection of M.vitis in mixed nematode populations and the detection of two J_(2)s or one male in 0.5 g soil samples,which will provide technical support for the investigation of the occurrence and damage of M.vitis and the formulation of efficient green co ntrol strategies.

Distribution of Virulence-Associated Genes of Avian Pathogenic Escherichia coli Isolates in China

216 avian pathogenic Escherichia coli （APEC） isolates were obtained from poultry with colibacillosis in different areas of China. Among them, 195 were serotyped as 078, 088, and 093. Thirteen virulence-associated genes, including fimC, iucD, iss, tsh, fyuA, irp2, eaeA, hlyE, colV, papC, stx2f, vat, and astA, were submitted to PCR amplification. The fimC gene was the most prevalent with a detection rate of 93.6%, followed by iucD （70.8%）, iss （58.8%）, and tsh （51.4%） in APEC isolates. The detection rate of high pathogenicity islands （HPI）-associatedfyuA and irp2 genes were both 44.9%, with no LEE （the locus of enterocyte effacement） island-associated gene eaeA detected. In terms of distribution patterns of the 13 virulence-associated genes, 5 isolates harborbed 10 genes, 19 isolates contained onlyfimC gene, and only 4 isolates had no virulence-associated gene detected. Different correlations of the virulence-associated genes with O serotypes were also investigated and 50% 078 isolates had a gene distribution patterns of fimC^＋iucD^＋irp2^＋fyuA^＋iss^＋colV^＋tsh^＋.

Genetic Diversity of Fusarium solani f. sp. cucurbitae, the Causal Root and Crown Rot of Cucurbits (Melon) by Using Molecular Markers and Control

Detection of F. solani f. sp. cucurbitae causal agent of the crown and root rot disease of melon race 1, race 2 is difficult. It is based only on morphological characteristic. In this study, forty isolates identified as Fusarium solani based on morphological characterization, F. solani was one of the most frequently isolated species. Molecular identification of these isolates by PCR technique using species-specific primer, indicated that thirty-two isolates, amplified product 580 bp (race 1) and two isolate amplified product 580 bp (race 2), while six isolates were not amplified with primer of both races. Production of Trichothecenes (T2-toxen, DON.) by Fusarium solani was conducted on isolates confirmed as belonging in the F. solani by PCR. The results indicated that the presence of Tri5, Tri13 genes is coding the ability of synthesis mycotoxin. In vitro, the results indicated that NPs (AgNPs, MgNPs) and chemical (Phylex) possess the antifungal properties against at various level. Treatment with (AgNPs 150 ppm, MgNPs 2%, 3% ppm) and 3% Phylex resulted in maximum inhabitation of F. solani . In vivo, five characters (height plant, hoot ant root fresh and dry weight) were measured based on the greenhouse, field experimental results. Treatment with (AgNPs, MgNPs) and Phylex had higher measured parameters than positive control.

HBV genotype characterization and distribution in patients with HBV-related liver diseases in Zhejiang Province, P. R. China: possible association of co-infection with disease prevalence and severity

BACKGROUND: There are 8 well-documented genotypes of hepatitis B virus (HBV) at this time point. Genotyping can be accomplished based on a partial sequence of hepatitis B virus (HBV) genome such as the pre-S or S gene. Several methods have been developed and used for HBV genotyping including direct sequencing, restriction fragment length polymorphism, line probe assay and enzyme-linked immunoassay. Recently, a novel, rapid and cost-effective genotyping method based on PCR amplification assay using type-specific primers that can identify all six major genotypes has been developed. This study was undertaken to characterise HBV genotypes and investigate the association between the prevalence of different genotypes and the severity of HBV-induced liver diseases. METHODS: Serum samples from carriers of HBV and patients with HBV-related liver diseases from Zhejiang Province were screened for viral serological markers using commercially available radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) kits. Serum HBV DNA load was determined by real-time detection PCR. A type-specific primer based the nested-PCR method was employed in the HBV genotyping. The genotype results obtained were confirmed by direct sequencing of nested PCR amplicons of the pre-S region. Ten samples of each genotype (B and C) were sequenced. RESULTS: The survey on a cohort of 125 HBV carriers in and around Hangzhou City, Zhejiang Province showed the existence of HBV genotypes A (0.8%), B (48%), C (40.8%), D (0.8%), mixed B and C (9.6%) and an absence of E and F genotypes. Distribution of HBV genotypes in patients with liver diseases revealed a statistically insignificant higher prevalence of genotype B in mild chronic hepatitis (CH). Among the three genotypes B, C and mixed B/C infections 11 (73.3%), 3 (20%) and 1 (6.7%), (P< 0.05), respectively in subjects with moderate CH, genotype B was significantly predominant. The infection patterns for genotypes B, C and B/C mixed in (i) liver cirrhosis (LC) 4 (23.5%), 10 (58.8%) and3 (17.7%) and (ii) hepatocellular carcinoma (HCC) 2 (28.6%), 5(71.4%) and 0 (0.0%) respectively revealed a marked association of C genotype with liver disease; however, the association was statistically insignificant (P >0.05). Differences in positive rate of HBeAg for the three genotypes B, 16(30.8%), C, 27(51.9%), and mixed B/C, 9(17.3%) were significant (P < 0. 05 ) , with genotype C showing predominance. CONCLUSIONS : These findings show an interesting distribution of HBV A-D genotypes in Zhejiang Province. Furthermore, our results indicate a novel and markedly high prevalence of mixed B/C genotype infections in subjects with severe CH and LC, and a possible association of mixed B/C infections with the severity of liver diseases in this region of China's Mainland.

A FIASCO-Based Approach for Detection and Diagnosis of Puccinia graminis f.sp.tritici in China

Stem or black rust of wheat, caused by the fungus Puccinia graminis Pers. f. sp. tritici Eriks.＆E. Henn. （Pgt）, has historically caused severe losses to wheat （Triticum aestivum L.） production worldwide. In the Fujian and Guangdong provinces of China, six moderate-to-severe epidemics of wheat stem rust have occurred, which caused destructive losses of wheat between 1949 and 1966, although these were brought under control by integrated management. A rapid and reliable detection of the pathogen will contribute to the accurate forecast and seasonal control of this disease. The objective of this study was to develop a diagnostic molecular marker generated from simple sequence repeats （SSR） for the early rapid identiifcation of P. graminis. The genomic DNA of P. graminis, Puccinia striiformis, Puccinia triticina and seven other species was ampliifed by a pair of SSR primers generated by the FIASCO （fast isolation by AFLP sequences containing repeats） enrichment protocol. The primer set Pgtw （f）/Pgtw （r） generated a polymorphic pattern displaying a 330-bp DNA fragment speciifc for P. graminis whereas no DNA fragment was obtained from other non-target wheat fungal pathogens. The detection limit of the primer was 1 ng DNA in a 25-mL PCR reaction. The SSR markers of P. graminis can also be used to detect the presence of latent hyphae in Pgt-infected wheat leaves as early as 30 h post-inoculation. A rapid approach to distinguish P. graminis from similar pathogenic fungi would be anticipated in further study.

Identification and Characterization of Putative Virulent Genes in Streptococcus equi ssp. zooepidemicus

Suppression subtractive hybridization （SSH） was performed with virulent strain ATCC35246 and avirulent strain ST171 to identify novel genes associated with virulence in Streptococcus equi ssp. zooepidemicus （SEZ）. There were fourteen genomic regions that only presented in virulent strain ATCC35246. These regions encoded 14 proteins, some of them were homologous to proteins associated with cellular surface structure, molecular synthesis, energy metabolism, regulation, transport systems, and other unknown functions. Primers for 6 particular regions were designed from the already published SEZ sequence. Then, we used PCR to evaluate the distribution and conservation of these 6 DNA fragments in various SEZ strains collected from different sources, regions, groups, and times. The results showed that these 6 DNA fragments were widely distributed in SEZ strains, yet they were not existence in the avirulent strain ST171. Moreover, these fragments could not be detected in other Streptococcus groups.

Study on the Genetic Transformation Conditions of Begonia wallichiana L.with Leaf Disc Method and Corresponding Identification Techniques

[Objectives]This study was conducted to improve the Agrobacterium-mediated genetic transformation system of Begonia wallichiana.[Methods]With sterilized tube seedling leaves as the recipient material and GFP as the reporter gene,optimization experiments were carried out in terms of infection time and method,co-cultivation time and method,and PCR detection technology.[Results]The transformation effect was better under the conditions of shaking Agrobacterium liquid,infection time of 1-2 h,and co-cultivation on sterilized filter paper for 2 d.After co-cultivation,the recipient material was first subjected to recovery culture,and then used for Hyg gradient screening,which was conducive to obtaining resistant transformants.The designed specific PCR detection technology could quickly identify false positives in resistant regenerated plants,and the proportion of transgenic plants was 16.7%.[Conclusions]The research results provide a new technical reference for the genetic transformation of ornamental plants.

Detection and Identification of Sugarcane Pokkah Boeng Pathogens of Main Sugarcane Varieties in Yunnan Province

[Objectives]This study was conducted to identify the pathogen species and dominant species of sugarcane pokkah boeng in main varieties in Yunnan sugarcane areas,so as to promote the healthy and sustainable development of sugarcane industry.[Methods]Specific primers Fv-F3/Fv-R3 and Fp-F4/Fp-R4 were designed based on the ribosomal DNA non-internal transcribed spacer(rDNA-ITS)gene sequences of Fusarium verticillioides and Fusarium proliferarum,the main pathogens of sugarcane pokkah boeng,and 117 typical sugarcane pokkah boeng samples collected from main varieties in different sugarcane areas of Yunnan Province were detected and analyzed by PCR.[Results]Among the 117 sugarcane pokkah boeng samples,112 samples were detected with F.verticillioides with a positive detection rate of 95.7%;103 samples were detected with F.proliferarum with a positive detection rate of 88%;103 samples were infected by F.verticillioides+F.proliferarum,and the compound infection rate was 88%;and the two pathogens were not detected in 5 samples,which might be sugarcane pokkah boeng caused by other species.PCR amplification products of 23 F.verticillioides positive samples and 19 F.proliferarum positive samples from different sugarcane varieties in different sugarcane areas were sequenced.The BLAST alignment results showed that the sequences of the 23 amplification products of F.verticillioides shared 99.45%-100.00%homology with F.verticillioides(GenBank accession number:KU508286),and the sequences of the 19 amplification products of F.proliferarum shared 99.26%-100.00%homology with the sequence of F.proliferarum(GenBank accession number:MK252904).Part of the F.verticillioids and 11 F.proliferarum sequences were selected to construct a phylogenetic tree,and the phylogenetic analysis showed that they belonged to the F.verticillioids group and F.proliferarum group,respectively.The results showed that F.verticillioides and F.proliferarum were the main pathogens causing sugarcane pokkah boeng of the main sugarcane varieties in Yunnan,and there was a common phenomenon of compound infection.F.verticillioides was the dominant species in Pu er,Lincang,Honghe and Yuxi sugarcane areas,but the detection rate of F.proliferarum was also high,and there were other species.In the future,the discovery of resistant germplasm resources and breeding of resistant varieties should be carried out aiming at these two pathogens of sugarcane pokkah boeng.[Conclusions]The study provides technical support for rapid identification of sugarcane pokkah boeng pathogens,and scientific basis for breeding resistant varieties and scientific disease prevention and control.

Detection of HBV DNA in Extrahepatic Tissue with PCR Comparison with Immunohistochemistry and in Situ Hybridization

Formalin-fixed paraffin-embedded tissue,including gallbladder,kidney,spleen,adrenal gland,heart,testicle,pancreas,and liver from 18 autopsied cases with HBV infection were studied with nested polymerase chain reaction

Establishment and evaluation based of a RIG-G gene detection system by TaqMan-MGB probe real-time PCR

Objective To establish a Taq Man-MGB fluorescent probe characterized real-time polymerase chain reaction(q PCR)method for detecting retinoic acid induced genes G(RIG-G)in human acute promyelocytic leukemia(M3).Analyze RIG-G expression levels in peripheral blood of both normal persons and M3 patients and

Strongyloides stercoralis prevalence and diagnostics in Vientiane, Lao People’s Democratic Republic

Background:Despite the high prevalence of strongyloidiasis in the Laotian population,Laotian hospitals still lack diagnostic capacity to appropriately diagnose Strongyloides stercoralis infections.This cross-sectional hospital-based study was conducted to assess the prevalence of Strongyloides stercoralis infection among hospitalized patients treated at Mahosot Hospital,the primary reference hospital of Lao People’s Democratic Republic(Lao PDR),and to validate feasible methods for diagnosing S.stercoralis infection at hospital’s laboratory.Methods:Between September and December 2018,stool samples of 104 inpatients were investigated for S.stercoralis infection by wet smear,Baermann technique,Koga Agar plate culture(KAPC),and real-time detection polymerase chain reaction(RTD-PCR)at the Infectious Diseases Ward of the Mahosot Hospital in Vientiane.The sensitivity,the specificity,the negative predictive value(NPV)of each diagnostic test,as well as their combination(s)was calculated using a composite reference standard(CRS).The correlation of the different test methods was assessed by chi-square or Fisher’s exact test.Cohen’s kappa coefficient was used to assess the diagnostic agreement of the different test methods.Results:The overall prevalence of S.stercoralis infections among the study population was 33.4%.The cumulative infection prevalence statistically significantly increased from the lowest age group of 40 years and below(22.4%),to the medium(40.0%)and to the oldest age group of 61 year and above(72.7%)(P=0.003).The cumulative infection prevalence of CRS was considerably higher in male(40.4%)compared to female patients(28.1%),but not statistically different(P=0.184).The diagnostic sensitivity of Baermann technique,KAPC,RTD-PCR,and the combination of Baermann technique and KAPC were 60.0,60.0,74.3,and 77.1%,respectively.Only 13 patients(37.1%)of the total 35 S.stercoralis patients diagnosed with any technique had a simultaneously positive diagnostic test with Baermann,KAPC and RTD-PCR.Conclusions:We identified Baermann technique and KAPC to be currently the most feasible and implementable standard methods for diagnosing S.stercoralis at a hospital setting such as Mahosot Hospital and provincial and district hospitals in Lao PDR and other low-and middle income countries in Southeast Asia.Trial registration:This study was approved by the National Ethics Committee for Health Research in Lao PDR(reference no.083/NECHR)and by the Ethics Committee Northwest and Central Switzerland(reference no.2018–00594).