A multiplex real-time RT-PCR assay for the detection of H1, H2 and H3 subtype avian influenza viruses.

Avian influenza viruses (AIVs) are influenza A viruses, of which subtypes H1, H2 and H3 are highly transmissible in poultry and have the risk of transmission to human as well. It is important to establish an accurate, sensitive and convenient means of virus detection. In this study, we developed a multiplex real-time RT-PCR assay based on conserved sequences of the virus hemagglutinin and matrix, and designed primers and probes for the simultaneous and rapid detection of AIV subtypes H1, H2 and H3. We used different subtypes of AIVs and other avian respiratory viruses for evaluation of the specificity of this method. The results showed good sensitivity, specificity and reproducibility. The detection limit was 10-100 copies per reaction. The method also achieved good concordance with the virus isolation method when compared to 81 poultry samples evaluated. It provides a new method for detecting mixed infections of AIVs.

Analysis of differential expression profile of miRNA in peripheral blood of patients with lung cancer.

PURPOSE: To identify potential molecular targets for lung cancer intervention and diagnosis, we analyzed the differential miRNA expression of peripheral blood between lung cancer patients and healthy controls. METHODS: Three pairs of cases' and controls' peripheral blood samples were evaluated for miRNA expression by microarray. 12 miRNAs were selected for RT-PCR validation and target genes prediction. In addition, 4 miRNAs were selected for future validation by RT-PCR in a large sample of 145 cases and 55 frequency-matched healthy controls. RESULTS: A total of 338 differentially expressed miRNAs were screened and identified by microarray. According to the fold changes, the top ten upregulated miRNAs were hsa-miR-124-3p, hsa-miR-379-5p, hsa-miR-3655, hsa-miR-450b-5p, hsa-miR-29a-5p, hsa-miR-200a-3p, hsa-miR-542-3p, hsa-miR-138-5p, hsa-miR-219a-2-3p, and hsa-miR-4701-3p, and the top ten downregulated miRNAs were hsa-miR-34c-5p, hsa-miR-135a-5p, hsa-miR-132-3p, hsa-miR-3178, hsa-miR-4449, hsa-miR-4999-3p, hsa-miR-1246, hsa-miR-4424, hsa-miR-1252-5p, and hsa-miR-24-2-5p. RT-PCR verification of the 12 miRNAs revealed that 5 of 8 upregulated miRNAs, 2 of 4 downregulated miRNAs showed a significant difference between the cases and controls (P < .05). A large number of target genes and their functional set showed overlapping among the 453 predicted target genes of the 12 miRNAs (P < .01). RT-PCR in the large sample confirmed the significant differential expression level of hsa-miR-29a-5p, hsa-miR-135a-5p, hsa-miR-542-3p, and hsa-miR-4491 between cases and controls (P < .05), and three of these microRNA, except hsa-miR-29a-5p, were significant after Bonferroni correction for adjustment of multiple comparisons. CONCLUSION: There was a significant difference in miRNAs expression in the peripheral blood between lung cancer patients and healthy controls, and 4 miRNAs were validated by a large-size sample.

[Effect of emodin on gut microbiota of rats with acute kidney failure].

The aim of this paper was to investigate the effect of emodin on gut microbiota in acute kidney injury rats( AKI). Rats were randomly divided into several groups: normal group,model group,low-dose of emodin group( 10 mg·kg~(-1)),medium-dose of emodin group( 25 mg·kg~(-1)),high-dose of emodin group( 50 mg·kg~(-1)) and control group( 5 mg·kg~(-1) of benazepril hydrochloride).The AKI model rats were established by intraperitoneal injection of small dose of gentamicin sulfate for 7 days. Two hours after intraperitoneal injection,except for the normal group and the model group,the other groups were given corresponding doses of drugs for 15 days. The serum levels of serum creatinine( SCr),urea nitrogen( BUN),plasma endotoxin level,24 h urinary protein and D-lactate in the plasma were determined by sarcosine oxidase,urease method,tal reagent method,bromo cresol chloroform method and double antibody sandwich enzyme-linked immunoadsorbent assay,respectively. Gut microbial communities were assayed by fluorescent quantitative PCR methods. HE staining was used to detect the pathological changes of the kidneys. Compared with the normal group,there were significant differences in body weight,urinary protein( UTP),bacterial endotoxin,urea nitrogen,creatinine,D-lactate in the plasma and four bacterial contents in the model group( P<0. 05). The urinary protein,urea nitrogen,D-lactate,creatinine and plasma bacterial endotoxin in control group and each emodin group were lower than those in model group,especially for high-dose of emodin( P<0. 01). Moreover,pathology resolution in high-dose emodin was better than other groups. Except for low-dose of emodin group,qRT-PCR data suggested that the amounts of Escherichia coli and Enterococcus in medication administration group were increased,while the amounts of Lactobacilli and Bifidobacterium were reduced compared with model group( P<0. 05),especially for high-dose of emodin( P<0. 01). There is a clear imbalance of gut microbiota in rats with AKI. Emodin could regulate the imbalance of gut microbiota,which might be one of the mechanisms of its effects on AKI rats.

Detection of Gallibacterium anatis by TaqMan fluorescent quantitative PCR.

To better understand the prevalence of Gallibacterium anatis in different poultry species, a rapid and accurate method was developed to detect G. anatis using a TaqMan fluorescent quantitative polymerase chain reaction (qPCR). Specific primers and a TaqMan probe were designed based on the reference gtxA gene sequence. The qPCR standard curve showed a good linear relationship, and the method showed good reproducibility, sensitivity, and specificity, indicating its suitability for G. anatis identification and quantitative analysis. A comparison of the detection results in 160 clinical swab samples showed that the detection rate (54.4%) of the qPCR for G. anatis was better than that of two conventional methods: gyrB gene-based qPCR for G. anatis (51.9%) and culture-based identification (34.4%). G. anatis was detected in layer chicken (77.3%), Silkie chicken (72.7%), and duck (27.1%) with relatively high detection rates, whereas dove (8.8%) and quail (3.0%) showed lower detection rates, indicating the different prevalence of G. anatis in different fowl species.

[Analyzing Colonization of Bifidobacteria in Infants with Real-time Fluorescent Quantitative PCR].

OBJECTIVES: In order to know how intestinal Bifidobacteria community could be built in the infants and whether the environmental factors could affect them, the present study was conducted to characterizethe species composition and trace the quantitative changes of intestinal Bifidobacteria of the infants in their early stages with non-culture dependent molecular method. The possible association of Bifidobacteria community of the infants with their health was also discussed. METHODS: Total 16 of full-term newborn infants born between March and April 2013 were recruited for the present study. Fecal samples were collected from them at 1 day, 2 days, 4 days, 7 days, 10 days, 14 days, 28 days, 3 months, 6 months and 1 year after birth. Real-time fluorescent quantitative PCR with genus and species specific premiers was used to detect Bifidobacteria and 8 predominate species in human intestine qualitatively and quantitatively present in these collected fecal samples. RESULTS: Total 136 fecal sample were collected and Bifidobacteria were detected from 93.4% (127/136) of them with the concentration of 1.0×10 5 to 1.0×10 11 CFU/g. Bifidobacteria were found in 83.3% of the fecal samples collected from the first day after birth with more than about 10 5 CFU/g. However, Bifidobacteria were detected relative low until 14 days and were taxonomically belonged only to one or two species. Bifidobacteria were found in almost 100% of the fecal samples collected after birth 28 days with more than 108 CFU/g, and the detected species of Bifidobacteria was increased to 3 species after 28 days to 6 months. All of the fecal samples collected from one year had more than 3 species of Bifidobacteria with high cell counts. Among the detected Bifidobacteria were B.breve 92.1%, B.infantis 66.1%, B. catenulatum 59.8%, B. bifidum 25.2%, B. longum 24.4%, B.dentium 13.4%, B.angulatum 5.5% and B.adolescentis 1.6%, respectively. CONCLUSIONS: The detected Bifidobacteria greatly varied qualitatively and quantitatively after birth to one year which could be considered as the important and sensitive period for Bifidobacteria to colonize and built its communityin the infants. Different from previous studies, the colonization of Bifidobacteria in the tested infants was found delayed and the composition and diversity of Bifidobacteria species was different from other studies. These might result from different deliveryway, feeding pattern and other environmental factors related to the tested infants.

[Correlation between RNA Expression Level and Early PMI in Human Brain Tissue].

OBJECTIVES: To explore the correlation between the expression levels of several RNA markers in human brain tissue and early postmortem interval （PMI）. METHODS: Twelve individuals with known PMI （range from 4.3 to 22.5 h） were selected and total RNA was extracted from brain tissue. Eight commonly used RNA markers were chosen including ß-actin, GAPDH, RPS29, 18S rRNA, 5S rRNA, U6 snRNA, miRNA-9 and miRNA-125b, and the expression levels were detected in brain tissue by real-time fluorescent quantitative PCR. The internal reference markers with stable expression in early PMI were screened using geNorm software and the relationship between its expression level and some relevant factors such as age, gender and cause of death were analyzed. RNA markers normalized by internal reference were inserted into the mathematic model established by previous research for PMI estimation using R software. Model quality was judged by the error rate calculated with estimated PMI. RESULTS: 5S rRNA, miRNA-9 and miRNA-125b showed quite stable expression and their expression levels had no relation with age, gender and cause of death. The error rate of estimated PMI using ß-actin was 24.6%, while GAPDH was 41.0%. CONCLUSIONS: 5S rRNA, miRNA-9 and miRNA-125b are suitable as internal reference markers of human brain tissue owing to their stable expression in early PMI. The expression level of ß-actin correlates well with PMI, which can be used as an additional index for early PMI estimation.

Development of rapid nucleic acid testing techniques for common respiratory infectious diseases in the Chinese population.

Background: Most respiratory viruses can cause serious lower respiratory diseases at any age. Therefore, timely and accurate identification of respiratory viruses has become even more important. This study focused on the development of rapid nucleic acid testing techniques for common respiratory infectious diseases in the Chinese population. Methods: Multiplex fluorescent quantitative polymerase chain reaction (PCR) assays were developed and validated for the detection of respiratory pathogens including the novel coronavirus (SARS-CoV-2), influenza A virus (FluA), parainfluenza virus (PIV), and respiratory syncytial virus (RSV). Results: The assays demonstrated high specificity and sensitivity, allowing for the simultaneous detection of multiple pathogens in a single reaction. These techniques offer a rapid and reliable method for screening, diagnosis, and monitoring of respiratory pathogens. Conclusion: The implementation of these techniques might contribute to effective control and prevention measures, leading to improved patient care and public health outcomes in China. Further research and validation are needed to optimize and expand the application of these techniques to a wider range of respiratory pathogens and to enhance their utility in clinical and public health settings.

Analysis of three cases with false positive PCR results of non tuberculosis mycobacterium.

Background: Real-time fluorescent quantitative PCR (RT-PCR) can effectively distinguish between Mycobacterium tuberculosis (MTB) and Non-tuberculosis mycobacterium (NTM), but when there are overlapping sequences between other pathogens (such as Nocardia otidiscaviarum, Mycobacterium parantracellulare, Mycolicibacterium fluoranthenivorans) and NTM, abnormal amplification curves may appear. Case presentation: The clinical manifestations of the three patients were fever and respiratory symptoms. Chest CT showed "multiple lung infections". The acid-fast bacilli were negative by microscopic examination. The results of RT-PCR detection of Mycobacterium tuberculosis DNA showed that they are all NTM, while the results of DNA microarray method showed that there were no non-Mycobacterium tuberculosis. Identified by MALDI-TOF mass spectrometry, they are Nocardia otidiscaviarum, Mycobacterium parantracellale, Mycolicibacterium fluoranthenivorans. We found that the sequences of the above three bacteria can be combined with the primers and probes used for NTM PCR detection, resulting in false positive. Conclusions: In the RT-PCR detection of mycobacteria, if there's abnormal amplification, and the mycobacterial species cannot be identified, the amplified products sequencing or MALDI- TOF mass spectrometry identification will help avoid the omission of rare pathogens.

Distribution characteristics of antibiotic resistance genes and microbial diversity in the inshore aquaculture area of Wenchang, Hainan, China.

The rapid development of marine aquaculture has led to the increased use and release of antibiotics into the marine environment, consequently contributing to the emergence of antibiotic resistance. Information on antibiotic resistance in nearshore marine aquaculture areas remains limited, and research on the microbial composition and potential hosts of antibiotic resistance genes (ARGs) in marine aquaculture areas is scarce. This study used SmartChip real-time fluorescent quantitative PCR and qPCR to quantitatively analyze 44 ARGs and 10 mobile genetic elements (MGEs) genes in 12 sampling points in the nearshore aquaculture area of Wenchang. High-throughput sequencing of 16S rRNA was used to study microbial diversity in the study area, to clarify the correlation between ARGs, MGEs, and microbial diversity, and to determine the possible sources and potential hosts of ARGs. The results showed that a total of 37 ARGs and 8 MGEs were detected in the study area. The detection rate of 9 ARGs (aac(6')-Ib(aka aacA4)-02, catA1, cmlA, cfr, sul1, sul2, sulA/folP-01, tetC, tetX) was 100 %. The absolute abundance of ARGs in the 12 sampling points ranged from 2.75 × 107 to 3.79 × 1010 copies·L-1, and the absolute abundance of MGEs was 1.30 × 105 to 2.54 × 107 copies·L-1, which was relatively high compared to other research areas. ARGs and MGEs were significantly correlated, indicating that MGEs play an important role as a mediator in the spread of ARGs. At the phylum level, Proteobacteria and Cyanobacteria were the dominant bacteria in the study area, with HIMB11 and unidentifiedChloroplast being the dominant levels, respectively. Network analysis of ARGs and microorganisms (genus level) revealed that Cognatishimia, Thalassobius, Aestuariicoccus, Thalassotalea, and Vibrio were significantly correlated with multiple ARGs and were the main potential hosts of ARGs in the nearshore waters of Wenchang.

Investigation of porcine circovirus type 2 and porcine circovirus type 3 infections based on dual TaqMan fluorescent quantitative PCR method and genetic evolutionary analysis of these two viruses.

Introduction: Porcine circovirus type 2 (PCV2) is the pathogen of Porcine Circovirus Associated Diseases. Porcine circovirus type 3 (PCV3) is a novel porcine circovirus associated with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive failure. PCV2 is clearly pathogenic, while the pathogenicity of PCV3 remains controversial, so it is crucial to monitor the prevalence of PCV2 and PCV3 in healthy and diseased pigs to investigate the effects of PCV3 and PCV2 on the health status of pigs. Methods: Here, we developed a PCV2 and PCV3 dual TaqMan quantitative PCR (qPCR) method to test samples from healthy and diseased pigs, to clarify the differences in the positive rates and viral copy numbers of PCV2 and PCV3, and to analyze the genetic evolution and molecular characterization of the viral genomes obtained with sequence alignment and phylogenetic analysis, homology and structural analysis of Cap proteins, and selection pressure analysis. Results: We successfully established a dual TaqMan qPCR method for PCV2 and PCV3 with good repeatability, specificity and sensitivity. In total, 1,385 samples from 15 Chinese provinces were tested with the established qPCR. The total positive rates were 37.47% for PCV3 and 57.95% for PCV2, and the coinfection rate for was 25.49%. The positive rates of PCV3 and PCV2 in 372 healthy pigs were 15.05 and 69.89%, respectively, and the coinfection rate was 12.90%. The positive rates of PCV3 and PCV2 in 246 diseased pigs were 55.69 and 83.33%, respectively, and the coinfection rate was 47.97%. Eighteen PCV3 genomes and 64 PCV2 genomes were identified, including nine each of the PCV3a-1 and PCV3b genotypes, eight of PCV2a, 16 of PCV2b, and 40 of PCV2d. The amino acid identity within the PCV3 Cap proteins was 94.00-100.0%, whereas the PCV2 Cap proteins showed an identity of 81.30-100.0%. PCV3 Cap was most variable at amino acid sites 24, 27, 77, 104 and 150, whereas PCV2 Cap had 10-13 unique sites of variation between genotypes. Discussion: These results clarify the prevalence and variations of PCV2 and PCV3 in healthy and diseased pigs, which will provide a basis for the prevention and control of the two viral infections.

A duplex fluorescent quantitative PCR assay to distinguish the genotype I, II and I/II recombinant strains of African swine fever virus in China.

African swine fever (ASF) is a severe, hemorrhagic, and highly contagious disease caused by the African swine fever virus (ASFV) in both domestic pigs and wild boars. In China, ASFV has been present for over six years, with three genotypes of strains prevalent in field conditions: genotype I, genotype II, and genotype I/II recombinant strains. In order to differentiate among these three ASFV genotypes, a duplex fluorescent quantitative PCR method was established using specific probes and primers designed based on viral genes MGF_110-1L and O61R from ASFV strains reported in the GenBank database. Following optimization of reaction conditions, a duplex fluorescent quantitative PCR method was successfully developed. This method demonstrated no cross-reactivity with porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), classic swine fever virus (CSFV), porcine pseudorabies virus (PRV), porcine circovirus 2 (PCV2), porcine circovirus 3 (PCV3), highlighting its specificity. Sensitivity analysis revealed that the limits of detection (LODs) of this method were 2.95 × 10-1 copies/µL for the MGF_110-1L gene and 2.95 × 100 copies/µL for the O61R gene. The inter- and intra-group coefficients of variation were both <1%, indicating high reproducibility. In summary, the establishment of this duplex fluorescent quantitative PCR method not only addresses the identification of the ASFV recombinant strains but also allows for simultaneous identification of the three epidemic genotype strains.

Diagnostic Efficacy of Plasma-Based Real-Time PCR for Schistosomiasis Japonica in Mice before and after Treatment with Praziquantel.

The prevalence of schistosomiasis japonica in China is now characterized by a low epidemic rate and low-intensity infections. Some diagnostic methods with high sensitivity and specificity are urgently needed to better monitor this disease in the current situation. In this study, the detection efficacy of a real-time fluorescent quantitative PCR (qPCR) assay was assessed for schistosomiasis japonica in mice, and before and after treatment with praziquantel (PZQ). Our results showed that the sensitivity of the qPCR was 99.3% (152/153, 95% CI: 96.41-99.98%) and its specificity was 100% (77/77, 95% CI: 95.32-100%) in mice infected with different numbers of Schistosoma japonicum. After the oral administration of PZQ, mice infected with 10 cercariae or 40 cercariae were all Schistosoma japonicum-negative 6 weeks after treatment. However, the negativity rates on a soluble egg antigen (SEA)-based enzyme-linked immunosorbent assay (ELISA) were only 34.8% (8/23, 10 cercariae group) and 6.7% (1/15, 40 cercariae group) at the sixth week after PZQ treatment. These results demonstrated that the qPCR method had good sensitivity and specificity, and suggested that its sensitivity correlated with the infection intensity in mice. Moreover, this method had better potential utility for evaluating the treatment efficacy of PZQ in schistosome-infected mice than SEA-based ELISA.

[Establishment and evaluation of a method for extracting exogenous short DNA fragments of Schistosoma japonicum from urine samples].

OBJECTIVE: To establish the method for extracting exogenous short DNA fragments of Schistosoma japonicum from urine samples, and to evaluate the efficiency of this method for extraction from urine samples treated with various methods. METHODS: The S. japonicum SjG28 gene fragment was selected as a target sequence, and the 81 bp short DNA fragment was amplified on the target sequence using PCR assay. Following characterization using sequencing, the short DNA fragment was added into the urine samples as an exogenous short DNA fragment. Primers and probes were designed with SjG28 as a target gene, to establish the real-time fluorescent quantitative PCR (qPCR) assay. The sensitivity of this qPCR assay was evaluated with exogenous short DNA fragments that were diluted at a 1:10 dilution ratio as the DNA template, and the specificity of the qPCR assay was evaluated with the genomic DNA of S. mansoni, S. haematobium, Babesia, Ancyiostoma duodenaie, Cionorchis sinensis, and Paragonimus westermani as DNA templates. Exogenous short DNA fragments were added into artificial and healthy volunteers' urine samples, followed by pH adjustment, centrifugation and concentration, and the efficiency of extracting exogenous short DNA fragments from urine samples was compared with the QIAmp Viral RNA Mini Kit (Qiagen kit) and BIOG cfDNA easy kit (BIOG kit). RESULTS: An 81 bp small DNA fragment of S. japonicum was successfully prepared, and the lowest detection limit of the established qPCR assay was 100 copies/µL of the 81 bp small DNA fragment of S. japonicum. If the genomic DNA of S. japonicum, S. mansoni, S. haematobium, Babesia, A. duodenaie, C. sinensis, and P. westermani served as DNA templates, the qPCR assay only detected fluorescent signals with S. japonicum genomic DNA as the DNA template. If the pH values of artificial urine samples were adjusted to 5, 6, 7 and 8, the recovery rates were (49.12 ± 2.09)%, (84.52 ± 4.96)%, (89.38 ± 3.32)% and (87.82 ± 3.90)% for extracting the exogenous short DNA fragment of S. japonicum with the Qiagen kit, and were (2.30 ± 0.07)%, (8.11% ± 0.26)%, (13.35 ± 0.61)% and (20.82 ± 0.68)% with the BIOG kit, respectively (t = 38.702, 26.955, 39.042 and 29.571; all P values < 0.01). If the Qiagen kit was used for extracting the exogenous short DNA fragment from artificial urine samples, the lowest recovery rate was seen from urine samples with a pH value of 5 (all P values < 0.05), and there were no significant differences in the recovery rate from urine samples with pH values of 6, 7 and 8 (all P values > 0.05). Following centrifugation of artificial [(64.30 ± 1.00)% vs. (58.87 ± 0.26)%; t = 12.033, P < 0.05] and healthy volunteers' urine samples [(31 165 ± 1 017) copies/µL vs. (28 471 ± 818) copies/µL; t = 23.164, P < 0.05]. In addition, concentration of artificial urine samples with the 10 kDa Centrifugal Filter and concentration of healthy volunteers' urine samples with the 100 kDa Centrifugal Filter were both effective to increase the recovery of the Qiagen kit for extracting the exogenous short DNA fragment of S. japonicum (both P values < 0.01). CONCLUSIONS: A method for extracting exogenous short DNA fragments of S. japonicum from urine samples has been successfully established, and the Qiagen kit has a high extraction efficiency. Adjustment of urine pH to 6 to 8 and concentration of healthy volunteers' urine samples with the 100 kDa Centrifugal Filter are both effective to increase the efficiency of extracting exogenous short DNA fragments of S. japonicum.

A multiplex real-time fluorescence-based quantitative PCR assay for calf diarrhea viruses.

Introduction: Calf diarrhea is a significant condition that has a strong effect on the cattle industry, resulting in huge economic losses annually. Bovine torovirus (BToV), bovine enterovirus (BEV), bovine norovirus (BNoV), bovine coronavirus (BCoV), bovine rotavirus (BRV), and bovine viral diarrhea virus (BVDV) are key pathogens that have been implicated in calf diarrhea. Among these viruses, there remains limited research on BToV, BEV, and BNoV, with no available vaccines or drugs for their prevention and control. Although commercial vaccines exist for BCoV, BRV, and BVDV, the prevalence of these diseases remains high. Methods: To address this issue, we developed a multiplex real-time fluorescence quantitative PCR method for detecting BToV, BEV, BNoV, BCoV, BRV, and BVDV. This method can be used to effectively monitor the prevalence of these six viruses and serve as a reference for future prevention and control strategies. In this study, we specifically designed primers and probes for the BNoV Rdrp, BEV 5'UTR, BToV M, BCoV N, BRV NSP5, and BVDV 5'UTR genes. Results: This method was determined to be efficient, stable, and sensitive. The lowest detectable levels of plasmids for BNoV, BEV, BToV, BRV, BCoV, and BVDV were 1.91 copies/µL, 96.0 copies/µL, 12.8 copies/µL, 16.4 copies/µL, 18.2 copies/µL, and 65.3 copies/µL, respectively. Moreover, the coefficients of variation for all six detection methods were < 3%; they also exhibited a strong linear relationship (R2 ≥ 0.98), and an amplification efficiency of 90%-110%. A total of 295 fecal and anal swabs were collected from calves with diarrhea in Guangdong, China. The positive rates for BToV, BEV, BNoV, BCoV, BR, and BVDV were determined to be 0.34% (1/295), 6.10% (18/295), 0.68% (2/295), 1.36% (4/295), 10.85% (32/295), and 2.03% (6/295), respectively. Notably, BEV and BRV exhibited the highest prevalence. Discussion: Additionally, this study identified the occurrence of BToV and BNoV in Guangdong for the first time. In summary, this study successfully established an effective method for detecting several important bovine viruses; ultimately, this holds strong implications for the future development of the cattle industry.

Selection and validation of reference genes for quantitative real-time PCR in Cymbidium sinense.

Selection of reference genes (RGs) is important for the accurate analysis of real-time quantitative PCR (RT-qPCR) results. This study screened RGs of Cymbidium sinense for more accurate quantification of target genes. The two most stable RGs for all tissues were ACT and EF1α, those for vegetative organs were UBQ3 and ACT, while those for reproductive organs as well as organs in the full flowering stage were EF1α and ACT. The AGAMOUS (CsAG1) expression level was verified using EF1α, ACT, GAPDH, UBQ2 and UBQ3 as RG. The expression profile of CsAG1 was consistent when normalized with EF1α, ACT and UBQ3. The results have practical value for the expression of key genes during the development of C. sinense.

A duplex fluorescent quantitative PCR assay to distinguish the genotype I and II strains of African swine fever virus in Chinese epidemic strains.

African swine fever (ASF) is a highly contagious hemorrhagic disease that affects domestic and wild pigs. A recent study reported that both ASF virus (ASFV) genotypes I and II have invaded farm-raised pigs in China, causing chronic infection and morbidity. To develop a duplex fluorescent quantitative PCR method to distinguish the ASFV genotypes I and II in Chinese epidemic strains, the probes and primers were designed based on the B646L sequences of genotypes I and II listed in the GenBank database. After optimizing the system, a duplex fluorescent quantitative PCR method for simultaneous detection of ASFV genotypes I and II B646L genes was successfully established. This method had no cross-reaction with Porcine circovirus type 2 (PCV2), Pseudorabies virus (PRV), or Porcine Parvovirus (PPV), indicating that it has strong specificity. The sensitivity results indicated that the minimum detection limit of ASFV genotypes I and II B646L was 10 copies/Rxn. The inter- and intra-group coefficients of variation were both <3%, indicating that the method was highly reproducible. Therefore, the established duplex fluorescent quantitative PCR assay is important for the differential detection and epidemiological investigation of ASFV.

[In silico cloning, expression and bioinformatics analysis of StZnT11 in Solanum tuberosum].

Solanum tuberosum Zinc transporter 11 (StZnT11) is very important for maintaining zinc homeostasis in cells. The study on the expression of StZnT11 under abiotic stress and biotic stress laid a foundation for verifying the role of potato StZnT11 in the process of biotic stress of Ralstonia solanacearum species complex. According to the designated EST sequence, the homology of the original sequence was analyzed by using the Blast tool in NCBI, and a homologous object sequence with the highest similarity, coverage and e expectation value was selected. StZnT11 gene is obtained by Silico Cloning. The sequence and coding amino acid composition, physicochemical properties, molecular evolution, phosphorylation site and advanced structure of Solanum tuberosum StZnT11 gene were analyzed by bioinformatics method. The results showed that the cDNA gene is 1 300 bp in length, encoding a protein containing 348 amino acid residues, including 23 phosphorylation sites, one signal peptide and nine transmembrane regions, and is a hydrophobic protein located the plasma membrane. Through amino acid sequence alignment, StZnT11 protein has a high homology with zinc transporter from tobacco, tomato, pepper and other plants. The results of real-time fluorescence quantitative polymerase chain reaction showed that, StZnT11 is up-regulated by different concentrations of exogenous plant hormone abscisic acid (ABA). Tissue localization showed that StZnT11 was mainly expressed in specific tissues (phloem and leaf vascular bundles of stem vascular system). These results provide a theoretical basis for further experimental cloning and functional verification of the gene.

Research progress in molecular biology related quantitative methods of MicroRNA.

MicroRNAs (miRNAs) are small RNAs of 18-25 nucleotides in length that are widely distributed in eukaryotes and are produced by DNA transcription. As regulators of post-transcriptional gene expression, it plays an important role in the physiological processes of cells. As some miRNAs in the body are abnormally expressed at different and earlier stages of diseases, this phenomenon suggests that accurate, sensitive and specifical detection of them can be helpful for early and differential diagnosis. To expound the technological progress of miRNA detection, we reviewed all the related articles in PubMed database published before May 6, 2019, with the following keywords: "miRNA", "real-time fluorescent quantitative PCR", "electrochemical detection", "next-generation sequencing", "digital PCR technology". Original articles and reviews on the topics were selected. The present methods established for quantitative detection of miRNAs mainly relies on various probe design and labeling techniques, and the improvement of the sensitivity and specificity of detection is often through combination of microarray chips, real-time fluorescent quantitative PCR, high-throughput sequencing and other techniques. This paper combines the existing microRNA detection methods to provide a reference for researchers to choose the best detection method.

Molecular and Toxicity Analyses of White Granulated Sugar and Other Processing Products Derived From Transgenic Sugarcane.

This study aimed to prepare the sugar industry for the possible introduction of genetically modified (GM) sugarcane and derived retail sugar products and to address several potential public concerns regarding the characteristics and safety of these products. GM sugarcane lines with integrated Cry1Ab and EPSPS foreign genes were used for GM sugar production. Traditional PCR, real-time fluorescent quantitative PCR (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) were performed in analyzing leaves, stems, and other derived materials during sugar production, such as fibers, clarified juices, filter mud, syrups, molasses, and final GM sugar product. The toxicity of GM sugar was examined with a feeding bioassay using Helicoverpa armigera larvae. PCR and RT-qPCR results showed that the leaves, stems, fibers, juices, syrups, filter mud, molasses, and white granulated sugar from GM sugarcane can be distinguished from those derived from non-GM sugarcane. The RT-qPCR detection method using short amplified product primers was more accurate than the traditional PCR method. Molecular analysis results indicated that trace amounts of DNA residues remain in GM sugar, and thus it can be accurately characterized using molecular analysis methods. ELISA results showed that only the leaves, stems, fibers, and juices sampled from the GM sugarcane differed from those derived from the non-GM sugarcane, indicating that filter mud, syrup, molasses, and white sugar did not contain detectable Cry1Ab and EPSPS proteins. Toxicity analysis showed that the GM sugar was not toxic to the H. armigera larvae. The final results showed that the GM sugar had no active proteins despite containing trace amounts of DNA residues. This finding will help to pave the way for the commercialization of GM sugarcane and production of GM sugar.

Development and verification of multiplex fluorescent quantitative PCR for detection of bacteria and fungi in cell culture medium / 中国生物制品学杂志

@#Objective To develop and verify a multiplex fluorescent quantitative PCR method for detection of common bacterial and fungal contaminants in cell culture medium.Methods According to NUC gene of Staphylococcus aureus,COLA gene of Clostridium spore,ITS-2 segment sequence of Candida albicans,a set of primers and probes were designed for each respectively,and using UBI3 gene of capsicum introduced as external standard gene,a triple reaction system of Staphylococcus aureus,Clostridium spore and external standard gene and a double reaction system of Candida albicans and external standard gene were established.The primer specificity,linear range,limit of detection,specificity,anti-interference performance and precision of the method were verified.Finally,100 samples of 293T cell culture medium were detected by using the developed method,which was compared with the common PCR method.Results Three pairs of primers all amplified about 100 bp specific gene bands corresponding to the three strains at different annealing temperatures(56,57,58 and59 ℃),and the size was consistent with the expected.In the range of 5.80 × 10~6 — 5.80 × 10~2 copies/μL,the standard plasmids of the three strains showed a good linear relationship with the Ct values.The standard curve equations were:Y=-3.373 X+37.48,Y=-3.557X+36.59 and Y=-3.536 X+39.78,each R~2> 0.99,respectively,and the amplification efficiency was in the range of 90%—110%.All the limits of detection of the three strains were 10~1 CFU/mL.The primers and probes of the three strains showed no specific amplification on the genomic DNA of six kinds of cells that were prone to cross-reaction.The genomic DNA of 293T cells,Yeast,Escherichia coli and Mycoplasma sp.had no effect on the detection.The CVs of repeatability and intermediate precision verification were both less than 15%.Among 100 cell culture medium samples,14 positive and 86 negative samples were detected,and the results of common PCR method for three positive and two negative samples randomly selected were consistent with the developed method.Conclusion The multiplex fluorescent quantitative PCR method developed in this study for the detection of bacteria and fungi in cell culture medium has good specificity,anti-interference performance and precision,and is simple to operate with low cost and high sensitivity,which can quickly detect the contaminants during cell culture.