Gastrointestinal tract distribution of Salmonella enteritidis in orally in fected mice with a species-specific fluorescent quantitative polymerase chain reaction

AIM: To identify and understand the regular distribution pattern and primary penetration site for Salmonella enteritidis (S. enteritidis) in the gastrointestinal tract. METHODS: Based on the species-specific DNA sequence of S. enteritidis from GenBank, a species-specific real- time, fluorescence-based quantitative polymerase chain reaction (FQ-PCR) was developed for the detection of S. enteritidis. We used this assay to detect genomic DNA of S. enteritidis in the gastrointestinal tract, including duodenum, jejunum, ileum, cecum, colon, rectum, esophagus and stomach, from mice after oral infection. RESULTS: S. enteritidis was consistently detected in all segments of the gastrointestinal tract. The jejunum and ileum were positive at 8 h post inoculation, and the final organ to show a positive result was the stomach at 18 h post inoculation. The copy number of S. enteritidis DNA in each tissue reached a peak at 24-36 h post inoculation, with the jejunum, ileum and cecum containing high concentrations of S. enteritidis, whereas the duodenum, colon, rectum, stomach and esophagus had low concentrations. S. enteritidis began to decrease and vanished at 2 d post inoculation, but it was still present up to 5 d post inoculation in the jejunum, ileum andcecum, without causing apparent symptoms. By 5 d post inoculation, the cecum had significantly higher numbers of S. enteritidis than any of the other areas (P < 0.01), and this appeared to reflect its function as a repository for S. enteritidis. CONCLUSION: The results provided significant data for clarifying the pathogenic mechanism of S. enteritidis in the gastrointestinal tract, and showed that the jejunum, ileum and cecum are the primary sites of invasion in normal mice after oral infection. This study will help to further understanding of the mechanisms of action of S. enteritidis.

A quantitative polymerase chain reaction assay for the enumeration of brown tide algae Aureococcus anophagefferens in coastal waters of Qinhuangdao

Aureococcus anophagefferens, a small pelagophyte algae, has caused brown tide blooms in coastal waters of Qinhuangdao in recent years, presenting significant negative impacts on the shellfish mariculture industry. Under standard light microscopy, it is visually indistinguishable from other small algae in field samples due to its extremely small size. In this study, quantitative polymerase chain reaction（q PCR） based on 18 S r DNA sequences was developed and used to detect and enumerate A. anophagefferens. A linear regression（R2 = 0.91） was generated based on cycle thresholds value（Ct） versus known concentrations of A. anophagefferens. Twenty-two field samples collected in coastal waters of Qinhuangdao were subjected to DNA extraction and then analyzed using q PCR. Results showed that A. anophagefferens had a wide distribution in coastal waters along Qinhuangdao. Elevated A. anophagefferens abundance, category 3 brown tide blooms（〉200 000 cells/m L） occurred at Dongshan Beach and Tiger-stone Beach in August in 2013. In shellfish mariculture areas along coastal waters of Qinhuangdao, 4 stations had category 3 blooms, and 6 stations had category 2 blooms（35 000–200 000 cells/m L） in August and all stations had category 1 blooms（〉0 to ≤35 000 cells/m L） in October. Quantitative PCR allows for detection of A. anophagefferens cells at low levels in filed samples, which is essential to effective management and prediction of brown tide blooms.

Primary application of a real-time quantitative polymerase chain reaction for the detection of human breast cancer related novel gene-Metadherin expression

Objective:The aim of this study was to detect the expression level of Metadherin (MTDH) in peripheral blood of the breast cancer patients by real-time fluorescence quantitative polymerase chain reaction (PCR),and to explore the relationship between expression of Metadherin gene in the patients peripheral blood and the clinic-pathological features in breast cancer. Methods:Real-time fluorescence quantitative polymerase chain reaction was employed to determine the expression level of Metadherin gene in 80 peripheral blood samples of breast cancer patients and healthy donors. Results:The expression of Metadherin gene in breast cancer patients peripheral blood were positive,in which 34 breast cancer patients were highly expressed,accounting for 55.7%,while the expression of Metadherin gene in normal females peripheral blood were negative,there was statistical significance (Ratio = 2.02±0.81,P < 0.05); Ratio of the Metadherin expression in breast cancer patients peripheral blood and the glyceraldehyde-3-phosphate dehydrogenase expression was 1.15 ± 0.36. REST software analysis showed that the expression of Metadherin gene was significantly up-regulated in breast cancer. Conclusion:The SYBR Green I quantitative real-time polymerase chain reaction method can successfully detect the expression level of Metadherin gene. Expression level of Metadherin gene in breast cancer patients peripheral blood is closely related to survival,and it maybe involved in the development of breast cancer and used as an indicator of prognosis.

Confusing finding of quantitative fluorescent polymerase chain reaction analysis in invasive prenatal genetic diagnosis:A case report

BACKGROUND Quantitative fluorescent polymerase chain reaction(QF-PCR)is a rapid prenatal diagnostic method for abnormalities on chromosomes 21,18,and 13 and sex chromosomal aneuploidy.However,the value of QF-PCR in diagnosing chromosomal structural abnormalities is limited.In this article,we report a confusing QF-PCR finding in a pregnant woman who underwent amniocentesis.CASE SUMMARY The short tandem repeat marker AMXY(Xp22.2/Yp11.2)located on the sex chromosome exhibited a trisomic biallelic pattern,indicating that the karyotype of the fetus might be 47,XYY.Chromosome analysis performed on cultured amniocytes showed a normal male karyotype of the fetus.Copy number variation sequencing confirmed a 500 kb duplication at Yp11.2-Yp11.2(chrY:6610001_7110000)and a 250 kb duplication at Yp11.2-Yp11.2(chrY:7110001_7360000).CONCLUSION In conclusion,the comprehensive application of different methods could achieve a higher detection rate and accuracy for the prenatal diagnosis of chromosomal disorders through chromosomal testing.

Quantitative studies of the regular distribution pattern for Salmonella enteritidis in the internal organs of mice after oral challenge by a specific real-time polymerase chain reaction

AIM： To identify and understand the regular distribution pattern for Salmonella enteritidis （S. enteritidis） in the internal organs of mice after an oral challenge over a 3 wk period. METHODS： Assays based on the serovar-specific DNA sequence of S. enteritidis from GenBank, and a serovar-specific real-time, fluorescence-based quantitative polymerase chain reaction （FQ-PCR） were developed for the detection of S. enteritidis. We used this assay to detect genomic DNA of S. enteritidis in the blood and the internal organs, including heart, liver, spleen, kidney, pancreas, and gallbladder, from mice after oral challenge at different time points respectively.RESULTS： The results showed that the spleen was positive at 12 h post inoculation （PI）, and the blood was at 14 h PI. The organism was detected in the liver and heart at 16 h PI, the pancreas was positive at 20 h PI, and the final organs to show positive results were the kidney and gallbladder at 22 h PI. The copy number of S. enteritidis DNA in each tissue reached a peak at 24-36 h PI, with the liver and spleen containing high concentrations of S. enteritidis, whereas the blood, heart, kidney, pancreas, and gallbladder had low concentrations. S. enteritidis populations began to decrease and were not detectable at 3 d PI, but were still present up to 12 d PI in the gallbladder, 2 wk for the liver, and 3 wk for the spleen without causing apparent symptoms.CONCLUSION： The results provided significant data for understanding the life cycle of S. enteritidis in the internal organs, and showed that the liver and spleen may be the primary sites for setting itself up as a commensa over a long time after oral challenge. Interestingly, it may be the first time reported that the gallbladder is a site of carriage for S. enteritidis over a 12 d period. This study will help to understand the mechanisms of action of S. enteriCdis infection in vivo.

Micro-droplet Digital Polymerase Chain Reaction and Real-Time Quantitative Polymerase Chain Reaction Technologies Provide Highly Sensitive and Accurate Detection of Zika Virus

The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus(ZIKV)and in preventing serious neurological complications of ZIKV infection. In this study, we established micro-droplet digital polymerase chain reaction(ddPCR) and real-time quantitative PCR(RT-qPCR) protocols for the detection of ZIKV based on the amplification of the NS5 gene. For the ZIKV standard plasmid, the RT-qPCR results showed that the cycle threshold(Ct) value was linear from 10~1 to 10~8 copy/l L, with a standard curve R^2 of 0.999 and amplification efficiency of 92.203%;however, a concentration as low as 1 copy/l L could not be detected. In comparison with RT-qPCR, the dd PCR method resulted in a linear range of 10~1–10~4 copy/l L and was able to detect concentrations as low as 1 copy/l L. Thus, for detecting ZIKV from clinical samples, RT-qPCR is a better choice for high-concentration samples(above 10~1 copy/l L),while ddPCR has excellent accuracy and sensitivity for low-concentration samples. These results indicate that the ddPCR method should be of considerable use in the early diagnosis, laboratory study, and monitoring of ZIKV.

Detection of the Pandemic H1N1/2009 Influenza A Virus by a Highly Sensitive Quantitative Real-time Reverse-transcription Polymerase Chain Reaction Assay

A quantitative real time reverse-transcription polymerase chain reaction （qRT-PCR） assay with specific primers recommended by the World Health Organization （WHO） has been widely used successfully for detection and monitoring of the pandemic H1N1/2009 influenza A virus. In this study, we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus. The newly designed primers target three regions that are highly conserved among the hemagglutinin （HA） genes of the pandemic HlN1/2009 viruses and are different from those targeted by the WHO-recommended primers. The qRT-PCR assays with the newly designed primers are highly specific, and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human, swine, and raccoon dog origin. Furthermore, the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction, respectively. When tested with 83 clinical samples, 32 were detected to be positive using the qRT-PCR assays with our designed primers, while only 25 were positive by the assays with the WHO-recommended primers. These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic HIN1/2009 virus infection.

Correlation Between the Clinical Severity, Bacterial Load, and Inflammatory Reaction in Children with Mycoplasma Pneumoniae Pneumonia

Given the lack of defining features in the clinical manifestations and radiographic findings for children with mycoplasma pneumoniae pneumonia(MPP),quantitative polymerase chain reaction(qPCR)has become a useful diagnostic method.This study was performed to explore the relationship between the qPCR findings,clinical symptoms,and inflammatory markers in children with MPP.Four hundred children with MPP have been enrolled in this retrospective analysis.All clinical and analytical information,including mycoplasma pneumoniae(MP)PCR results,has been collected.Based on the PCR results,the patients were divided into groups with load values(copy number)<105(54 cases),2105 and<106(71 cases),2106 and<107(112 cases),>107 and<108(114 cases),and>108(49 cases).The clinical features(including symptoms and signs)and inflammatory indicators were compared among the groups.The incidence of high fever(above 39℃),thermal peak during the entire hospitalization period,fever duration,days of hospitalization,and plasma lactate dehydrogenase(LDH)levels were statistically correlated with the MP PCR load value in children with MPP.The analysis of relevance degree showed the correlative order as a thermal peak of hospitalization>duration of fever>period of hospitalization>LDH value>C-reactive protein value.The host immune response was significantly greater in the complication group than in the non-complication group.

Establishment of a new quantitative detection approach to adefovir-resistant HBV and its clinical application

AIM:To establish the more feasible and sensitive assessment approach to the detection of adefovir (ADV) resistance-associated hepatitis B virus (HBV) quasispecies.METHODS: Based on the characteristics of rtA181V/T and rtN236T mutations, a new approach based on real-time fluorescent quantitative polymerase chain reaction (RT-PCR) was established for the detection of ADV-resistant HBV quasispecies, total HBV DNA, rtA181 and rtN236 mutations in blood samples from 32 chronic hepatitis B (CHB) patients with unsatisfactory curative effect on ADV and compared with routine HBV DNA sequencing.RESULTS: Both the sensitivity and specificity of this new detection approach to ADV-resistant HBV quasispecies were 100%, which were much higher than those of direct HBV DNA sequencing. The approach was able to detect 0.1% of mutated strains in a total plasmid population. Among the 32 clinical patients, single rtA181 and rtN236T mutation and double rtA181T and rtN236T mutations were detected in 20 and 8, respectively, while ADV-resistant mutations in 6 (including, rtA181V/T mutation alone in 5 patients) and no associated mutations in 26.CONCLUSION: This new approach is more feasible and efficient to detect ADV-resistant mutants of HBV and ADV-resistant mutations before and during ADV treatment with a specificity of 100% and a sensitivity of 100%.

Quantitative analysis of a panel of gene expression in prostate cancer——with emphasis on NPY expression analysis

Objective： To investigate molecular alterations associating with prostate carcinoma progression and potentially provide information toward more accurate prognosis/diagnosis. Methods： A set of laser captured microdissected （LCM） specimens from 300 prostate cancer （PCa） patients undergoing radical prostatectomy （RP） were defined. Ten patients representing ＂aggressive＂ PCa, and 10 representing ＂non-aggressive＂ PCa were selected based on prostate-specific antigen （PSA） recurrence, Gleason score, pathological stage and tumor cell differentiation, with matched patient age and race between the two groups. Normal and neoplastic prostate epithelial cells were collected with LCM from frozen tissue slides obtained from the RP specimens. The expressions of a panel of genes, including NPY, PTEN, AR, AMACR, DD3, and GSTP1, were measured by quantitative real-time RT-PCR （TaqMan）, and correlation was analyzed with clinicopathological features. Results： The expressions of AMACR and DD3 were consistently up-regulated in cancer cells compared to benign prostate epithelial cells in all PCa patients, whereas GSTP1 expression was down regulated in each patient. NPY, PTEN and AR exhibited a striking difference in their expression patterns between aggressive and non-aggressive PCas （P=0.0203, 0.0284, and 0.0378, respectively, Wilcoxon rank sum test）. The lower expression of NPY showed association with ＂aggressive＂ PCas based on a larger PCa patient cohort analysis （P=0.0037, univariate generalized linear model （GLM） analysis）. Conclusion： Despite widely noted heterogeneous nature of PCa, gene expression alterations ofAM,4CR, DD3, and GSTP1 in LCM-derived PCa epithelial cells suggest for common underlying mechanisms in the initiation of PCa. Lower NPY expression level is significantly associated with more aggressive clinical behavior of PCa; PTEN and AR may have potential in defining PCa with aggressive clinical behavior. Studies along these lines have potential to define PCa-associated gene expression alterations and likely co-regulation of genes/pathways critical in the biology of PCa onset/progression.

The diagnostic significance of the detection of cytokeratin 19 mRNA by quantitative RT-PCR in benign and malignant pleural effusions

Objective: To evaluate the diagnostic significance of detecting cytokeratin 19 (CK19) mRNA by quantitative reverse transcription polymerase chain reaction (RT-PCR) in benign and malignant pleural effusions. Methods: CK19 mRNA was examined by quantitative RT-PCR and CK19 was detected by Enzyme-linked immunoadsorbent assay (ELISA) in 32 patients with malignant pleural effusions and 35 patients with benign pleural effusions. Results: On the threshold of 200 copies/μl, the positive rate of CK19 mRNA in patients with malignant pleural effusions was 62.5%. The positive rates of CK19 mRNA and CK19 in the malignant pleural effusions were significantly higher than those in the benign group (P<0.01). Furthermore, the positive rate of CK19 mRNA was higher than that of CK19 in the malignant group (P<0.05). Conclusion: Detection of CK19 mRNA can be a promising diagnostic marker in differential diagnosis of benign and malignant pleural effusions.

Rapid prenatal diagnosis of trisomy 21 by fluorescent quantitative multiplex polymerase chain reaction

Trisomy 21, also named Down syndrome was the most frequent autosomal aneuploidy and the most common cause of mental retardation. Fifty percent patients had congenital heart malformation. Every 20 minutes one case of trisomy 21 was born, and the incidence rate was 1 in 600 to 800 newborns in China.1 In two thirds of cases with trisomy 21, there was a spontaneous abortion, so the actual incidence was higher than that obtained postnatally.

Exploring the regulatory mechanism of tRNA-derived fragments 36 in acute pancreatitis based on small RNA sequencing and experiments

BACKGROUND Acute pancreatitis(AP)is a disease featuring acute inflammation of the pancreas and histological destruction of acinar cells.Approximately 20%of AP patients progress to moderately severe or severe pancreatitis,with a case fatality rate of up to 30%.However,a single indicator that can serve as the gold standard for prognostic prediction has not been discovered.Therefore,gaining deeper insights into the underlying mechanism of AP progression and the evolution of the disease and exploring effective biomarkers are important for early diagnosis,progression evaluation,and precise treatment of AP.AIM To determine the regulatory mechanisms of tRNA-derived fragments(tRFs)in AP based on small RNA sequencing and experiments.METHODS Small RNA sequencing and functional enrichment analyses were performed to identify key tRFs and the potential mechanisms in AP.Reverse transcription quantitative polymerase chain reaction(RT-qPCR)was conducted to determine tRF expression.AP cell and mouse models were created to investigate the role of tRF36 in AP progression.Lipase,amylase,and cytokine levels were assayed to examine AP progression.Ferritin expression,reactive oxygen species,malondialdehyde,and ferric ion levels were assayed to evaluate cellular ferroptosis.RNA pull down assays and methylated RNA immunoprecipitation were performed to explore the molecular mechanisms.RESULTS RT-qPCR results showed that tRF36 was significantly upregulated in the serum of AP patients,compared to healthy controls.Functional enrichment analysis indicated that target genes of tRF36 were involved in ferroptosisrelated pathways,including the Hippo signaling pathway and ion transport.Moreover,the occurrence of pancreatic cell ferroptosis was detected in AP cells and mouse models.The results of interference experiments and AP cell models suggested that tRF-36 could promote AP progression through the regulation of ferroptosis.Furthermore,ferroptosis gene microarray,database prediction,and immunoprecipitation suggested that tRF-36 accelerated the progression of AP by recruiting insulin-like growth factor 2 mRNA binding protein 3(IGF2BP3)to the p53 mRNA m6A modification site by binding to IGF2BP3,which enhanced p53 mRNA stability and promoted the ferroptosis of pancreatic follicle cells.CONCLUSION In conclusion,regulation of nuclear pre-mRNA domain-containing protein 1B promoted AP development by regulating the ferroptosis of pancreatic cells,thereby acting as a prospective therapeutic target for AP.In addition,this study provided a basis for understanding the regulatory mechanisms of tRFs in AP.

Correlations among core species corresponding to the clinical staging of periodontitis

The correlation between microbiota plays a vital role in the progression of periodontal disease.This study investigated the in situ interaction networks between periodontal pathogens in periodontal and peri-implant disease.We used quantitative real-time polymerase chain reaction and Pearson’s correlation coefficients to quantify the copy numbers and correlations of four oral core species—Fusobacterium nucleatum,Porphyromonas gingivalis,Prevotella intermedia,and Streptococcus gordonii—from 80 subgingival sites(healthy and with periodontitis or gingivitis)in patients with periodontitis,and 68 subgingival sites(healthy and with periodontitis,gingivitis,peri-implantitis,or peri-implant mucositis)in patients with implants.The highest bacterial counts were observed for Porphyromonas gingivalis and Prevotella intermedia at all the sites.Within the same cohorts,the bacterial loads were greater at diseased sites than at healthy sites.Bacterial counts did not differ among clinical sites in the same group(P>0.05)but differed between periodontitis and peri-implant mucositis sites in the two groups.Porphyromonas gingivalis,F.nucleatum,and Prevotella intermedia had strong correlations at gingivitis and healthy sites and moderate correlations at periodontitis sites in patients with periodontitis.In patients with implants,Prevotella intermedia,F.nucleatum,and S.gordonii had strong correlations only at peri-implantitis sites.Also,based on metagenomic analysis,F.nucleatum and Prevotella intermedia were significantly correlated at the subgingival plaque in peri-implantitis and periodontitis samples.Our results suggest that variations in microbe-microbe interactions in subgingival plaque reflect changes in the progression of periodontal disease,providing a new perspective for understanding the mechanisms of periodontitis and peri-implantitis.

An Evaluation Method of Human Gut Microbial Homeostasis by Testing Specific Fecal Microbiota

Research on microecology has been carried out with broad perspectives in recent decades,which has enabled a better understanding of the gut microbiota and its roles in human health and disease.It is of great significance to routinely acquire the status of the human gut microbiota;however,there is no method to evaluate the gut microbiome through small amounts of fecal microbes.In this study,we found ten predominant groups of gut bacteria that characterized the whole microbiome in the human gut from a large-sample Chinese cohort,constructed a real-time quantitative polymerase chain reaction(qPCR)method and developed a set of analytical approaches to detect these ten groups of predominant gut bacterial species with great maneuverability,efficiency,and quantitative features.Reference ranges for the ten predominant gut bacterial groups were established,and we found that the concentration and pairwise ratios of the ten predominant gut bacterial groups varied with age,indicating gut microbial dysbiosis.By comparing the detection results of liver cirrhosis(LC)patients with those of healthy control subjects,differences were then analyzed,and a classification model for the two groups was built by machine learning.Among the six established classification models,the model established by using the random forest algorithm achieved the highest area under the curve(AUC)value and sensitivity for predicting LC.This research enables easy,rapid,stable,and reliable testing and evaluation of the balance of the gut microbiota in the human body,which may contribute to clinical work.

Urine exosome mRNA-based test for monitoring kidney allograft rejection:Effects of sample transportation and storage,and interference substances

BACKGROUND Exosomes are 30-150 nm nanovesicles with sophisticated nucleic acids cargo,actively secreted by all cells within human body,and found in abundance in all body fluids,including urine.These extracellular vesicles have tremendous potential for next generation diagnostics,theoretically enabling noninvasive assessment of organ and tissue function via liquid biopsy analysis.AIM Recently,feasibility of an exosomal molecular test was demonstrated for postorgan transplant monitoring:Analysis of urine-derived exosomal mRNA cargo allowed early detection of kidney allograft rejection.Here,we further studied urine-derived exosomes and their mRNA content as a highly promising diagnostic modality.This included stability studies of urine samples and exosomal mRNA upon transportation from the point of collection to a centralized testing facility,short-term storage of urine at different conditions upon receipt till the point molecular assay is performed,and effects of various potentially interfering substances on the downstream quantitative polymerase chain reaction(qPCR)assay.METHODS The urine specimens were stored at various conditions and pre-processed in different ways.Next,samples were passed through the columns to capture all extracellular vesicles,the vesicles were lysed to release their content and the exosomal RNA was purified on the mini-columns,reverse transcription was performed,next pre-amplification,followed by a qPCR analysis for a panel of RESULTS To ensure exosomal RNA integrity,the harvested urine specimens should be shipped refrigerated,by overnight delivery.Urine can next be stored at the test site for up to 1 wk at 4°C,and long term should be frozen at-80°C.Urine specimens must be centrifuge at low G-force to deplete cells and debris,to ensure consistent top results in downstream molecular assays.All commonly used medications(tacrolimus,cyclosporin A,mycophenolic acid,everolimus,sirolimus,ascomycin,teriflunomide)were tested and confirmed that they do not cause assay interference.CONCLUSION mRNA from urine-derived exosomes was shown to be stable across a broad range of conditions and produced accurate results when analyzed via qPCR assay for detection of kidney allograft rejection.We identified the most optimal conditions for every step of the process,ensuring pre-analytical sample integrity and robust qPCR results.

RT-qPCR normalization of reference genes in different lifehistory stages of Gracilaria vermiculophylla(Rhodophyta)

The quantitative realtime polymerase chain reaction(RT-qPCR)is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization.We evaluated the expressions of 8 housekeeping genes:18S ribosomal rDNA(18S rDNA),28S ribosomal r DNA(28S rDNA),rubisco large subunit(rbc-L),β-actin(ACT),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),elongation factor 1(EF1),β-tubulin(Tub B),and P-phycoerythrin B(PEB),to select the suitable reference genes for different life-history stages(tetrasporophyte,carposporophyte,and male/female gametophyte)of Gracilaria vermiculophylla by absolute quantitative method.Softwares geNorm and BestKeeper were used to verify the results acquired from copy number analysis.Results show that the expression of identified reference genes varied in comparing groups composed of different type of life stages.It is suggested that 18S rDNA and TubB could be used for highly complex samples composed of mixed ploidy and phases.18S rDNA and 28S rDNA were also preferred for using among the matured isomorphic samples.But for samples with different maturities,TubB and ACT were recommended for tetrasporophytes and gametophytes respectively.

Prenatal diagnosis of Down syndrome using cell-free fetal DNA in amniotic fluid by quantitative fluorescent polymersase chain reaction

Backgroud Amniotic fluid （AF） supernatant contains cell-free fetal DNA （cffDNA） fragments.This study attempted to take advantage of cffDNA as a new material for prenatal diagnosis,which could be combined with simple quantitative fluorescent polymerase chain reaction （QF-PCR） to provide an ancillary method for the prenatal diagnosis of trisomy 21 syndrome.Methods AF supernatant samples were obtained from 27 women carrying euploid fetuses and 28 women carrying aneuploid fetuses with known cytogenetic karyotypes.Peripheral blood samples of the parents were collected at the same time.Short tandem repeat （STR） fragments on chromosome 21 were amplified by QF-PCR.Fetal condition and the parental source of the extra chromosome could be determined by the STR peaks.Results The sensitivity of the assay for the aneuploid was 93％ （26/28; confidence interval,CI：77％-98％） and the specificity was 100％ （26/26; CI：88％-100％）.The determination rate of the origin of the extra chromosome was 69％.The sensitivity and the specificity of the assay in the euploid were 100％ （27/27）.Conclusions Trisomy 21 can be prenatally diagnosed by the QF-PCR method in AF supernatant.This karyotype analysis method greatly reduces the requirement for the specimen size.It will be a benefit for early amniocentesis and could avoid pregnancy complications.The method may become an ancillary method for prenatal diagnosis of trisomy 21.

Association of Fusobacterium nucleatum infection with colorectal cancer in Chinese patients

AIM: To investigate Fusobacterium nucleatum (F. nucleatum) abundance in colorectal cancer (CRC) tissues and its association with CRC invasiveness in Chinese patients.METHODS: The resected cancer and adjacent normal tissues (10 cm beyond cancer margins) from 101 consecutive patients with CRC were collected. Fluorescent quantitative polymerase chain reaction (FQ-PCR) was applied to detect F. nucleatum in CRC and normal tissues. The difference of F. nucleatum abundance between cancer and normal tissues and the relationship of F. nucleatum abundance with clinical variables were evaluated. Fluorescence in situ hybridization (FISH) analysis was performed on 22 CRC tissues with the highest F. nucleatum abundance by FQ-PCR testing to confirm FQ-PCR results.RESULTS: The median abundance of F. nucleatum in CRC tissues [0.242 (0.178-0.276)] was significantly higher than that in normal controls [0.050 (0.023-0.067)] (P < 0.001). F. nucleatum was over-represented in 88/101 (87.1%) CRC samples. The abundance of F. nucleatum determined by 2-ΔCT was significantly greater in tumor samples [0.242 (0.178, 0.276)] than in normal controls [0.050 (0.023, 0.067)] (P < 0.001). The frequency of patients with lymph node metastases was higher in the over-abundance group [52/88 (59.1%)] than in the under-abundance group [0/13 (0%)] (P < 0.005). No significant association of F. nucleatum with other clinico-pathological variables was observed (P > 0.05). FISH analysis also found more F. nucleatum in CRC than in normal tissues (median number 6, 25th 3, 75th 10 vs 2, 25th 1, 75th 5) (P < 0.01).CONCLUSION: F. nucleatum was enriched in CRC tissues and associated with CRC development and metastasis.

Survivin isoforms and clinicopathological characteristics in colorectal adenocarcinomas using real-time qPCR

AIM:To investigate three isoforms of survivin in colorectal adenocarcinomas.METHODS:We used the LightCycler Technology(Roche),along with a common forward primer and reverse primers specific for the splice variants and two common hybridization probes labeled with fluorescein and LightCycler-Red fluorophore(LC-Red 640).Real time quantitative polymerase chain reaction(PCR) was performed on cDNAs from 52 tumor specimens from colorectal cancer patients and 10 unrelated normal colorectal tissues.In the patients group,carcinoembryonic antigen(CEA) and CA19-9 tumor markers were also measured immunochemically.RESULTS:Wild type survivin mRNA isoform was expressed in 48%of the 52 tumor samples,survivin-2b in 38%and survivin-ΔΕx3 in 29%,while no expression was found in normal tissues.The mRNA expression of wild type survivin presented a significant correlation with the expression of the ratio of survivin-2b,survivin-ΔΕx3,survivin-2b/wild type survivin and survivin-ΔΕx3/wild type survivin(P<0.001).The mRNA expression of wildsurvivin and survivin-ΔΕx3 was related with tumor size and invasion(P=0.006 and P<0.005,respectively).A significant difference was found between survivin-2b and morphologic cancer type.Also,the ratio of survivin-ΔEx3/ wild-survivin was significantly associated with prognosis.No association was observed between the three isoforms and grade,metastasis,Dukes stage and gender.The three isoforms were not correlated with CEA and CA19-9.CONCLUSION:Survivin isoforms may play a role in cell apoptosis and their quantification could provide information about clinical management of patients suffering from colorectal cancer.