Droplet digital polymerase chain reaction assay for methylated ring finger protein 180 in gastric cancer

BACKGROUND Gastric cancer(GC)is one of the most prevalent malignant tumors that endangers human health.Early diagnosis is essential for improving the prognosis and survival rate of GC patients.Ring finger protein 180(RNF180)is involved in the regulation of cell differentiation,proliferation,apoptosis,and tumorigenesis,and aberrant hypermethylation of CpG islands in the promoter is strongly associated with the occurrence and development of GC.Thus,methylated RNF180 can be used as a potential biomarker for GC diagnosis.AIM To use droplet digital polymerase chain reaction(ddPCR)to quantify the methylation level of the RN180 gene.A reproducible ddPCR assay to detect methylated RNF180 from trace DNA was designed and optimized.METHODS The primer and probe were designed and selected,the conversion time of bisulfite was optimized,the ddPCR system was adjusted by primer concentration,amplification temperature and amplification cycles,and the detection limit of ddPCR was determined.RESULTS The best conversion time for blood DNA was 2 h 10 min,and that for plasma DNA was 2 h 10 min and 2 h 30 min.The results of ddPCR were better when the amplification temperature was 56°C and the number of amplification cycles was 50.Primer concentrations showed little effect on the assay outcome.Therefore,the primer concentration could be adjusted according to the reaction system and DNA input.The assay required at least 0.1 ng of input DNA.CONCLUSION In summary,a ddPCR assay was established to detect methylated RNF180,which is expected to be a new diagnostic biomarker for GC.

Plasma exosomal hsa_circ_0079439 as a novel biomarker for early detection of gastric cancer

BACKGROUND Due to the poor prognosis of gastric cancer(GC),early detection methods are urgently needed.Plasma exosomal circular RNAs(circRNAs)have been suggested as novel biomarkers for GC.AIM To identify a novel biomarker for early detection of GC.METHODS Healthy donors(HDs)and GC patients diagnosed by pathology were recruited.Nine GC patients and three HDs were selected for exosomal whole-transcriptome RNA sequencing.The expression profiles of circRNAs were analyzed by bioinformatics methods and validated by droplet digital polymerase chain reaction.The expression levels and area under receiver operating characteristic curve values of plasma exosomal circRNAs and standard serum biomarkers were used to compare their diagnostic efficiency.RESULTS There were 303 participants,including 240 GC patients and 63 HDs,involved in the study.The expression levels of exosomal hsa_circ_0079439 were significantly higher in GC patients than in HDs(P<0.0001).However,the levels of standard serum biomarkers were similar between the two groups.The area under the curve value of exosomal hsa_circ_0079439 was higher than those of standard biomarkers,including carcinoembryonic antigen,carbohydrate antigen(CA)19-9,CA72-4,alpha-fetoprotein,and CA125(0.8595 vs 0.5862,0.5660,0.5360,0.5082,and 0.5018,respectively).The expression levels of exosomal hsa_circ_0079439 were significantly decreased after treatment(P<0.05).Moreover,the expression levels of exosomal hsa_circ_0079439 were obviously higher in early GC(EGC)patients than in HDs(P<0.0001).CONCLUSION Our results suggest that plasma exosomal hsa_circ_0079439 is upregulated in GC patients.Moreover,the levels of exosomal hsa_circ_0079439 could distinguish EGC and advanced GC patients from HDs.Therefore,plasma exosomal hsa_circ_0079439 might be a potential biomarker for the diagnosis of GC during both the early and late stages.

SMRT sequencing and ddPCR reveal the complexity of developmental trajectories and temporal dynamics of gut bifidobacterial communities in infants

Infant intestinal microbiome is closely linked with health and risk of disease. Bifidobacterium are important components of the infant gut and are known to confer various health effects on the host. However, few studies have described the precise composition and dynamics of early infant gut bifidobacterial communities. Thus, this was a pilot study aiming to describe the developmental trajectories and temporal dynamics of bifidobacterial communities in infants before 6 months of age. A total of 28 fecal samples from 4 infants(GF, ZZ, QM, TN, respectively)were collected and analyzed after 5, 15, 30, 60, 90, 120, 150, and 180 days of birth by a bifidobacteria-target method(based on single-molecule real-time sequencing of partial bifidobacterial rpsK genes)in conjunction with droplet digital polymerase chain reaction(ddPCR). The infant fecal microbiota comprised a total of 11 bifidobacterial species, including 4 major species, i.e., B. dentium(37.35%), B. catenulatum(32.04%), B. breve(22.24%), and B. animalis(8.02%). The infant microbiota showed highly individualized developmental trajectories. The leading species for GF was B. catenulatum, with a relatively stable developmental trajectory. In ZZ, B. breve was enriched, and the developmental trajectory was rather fluctuating. The most abundant species for QM and TN was B. dentium. The developmental trajectory of B. dentium in QM showed a trend of gradual decrease, whereas an opposite trend was seen in samples of TN. The results of ddPCR confirmed large variations in quantities of bifidobacteria between infants and suggested discordances in temporal dynamics of bifidobacterial communities during the first half year of infancy. In conclusion, our results suggested that the early infant gut bifidobacterial microbiota was highly complex and temporal dynamics, with individualized developmental trajectories, which should be considered in future research of infant gut microbiota.

Development of a droplet digital polymerase chain reaction assay for the sensitive detection of total and integrated HIV-1 DNA

Background:Total human immunodeficiency virus(HIV)DNA and integrated HIV DNA are widely used markers of HIV persistence.Droplet digital polymerase chain reaction(ddPCR)can be used for absolute quantification without needing a standard curve.Here,we developed duplex ddPCR assays to detect and quantify total HIV DNA and integrated HIV DNA.Methods:The limit of detection,dynamic ranges,sensitivity,and reproducibility were evaluated by plasmid constructs containing both the HIV long terminal repeat(LTR)and human CD3 gene(for total HIV DNA)and ACH-2 cells(for integrated HIV DNA).Forty-two cases on stable suppressive antiretroviral therapy(ART)were assayed in total HIV DNA and integrated HIV DNA.Correlation coefficient analysis was performed on the data related to DNA copies and cluster of differentiation 4 positive(CD4^(+))T-cell counts,CD8^(+)T-cell counts and CD4/CD8 T-cell ratio,respectively.The assay linear dynamic range and lower limit of detection(LLOD)were also assessed.Results:The assay could detect the presence of HIV-1 copies 100%at concentrations of 6.3 copies/reaction,and the estimated LLOD of the ddPCR assay was 4.4 HIV DNA copies/reaction(95%confidence intervals[CI]:3.6-6.5 copies/reaction)with linearity over a 5-log_(10)-unit range in total HIV DNA assay.For the integrated HIV DNA assay,the LLOD was 8.0 copies/reaction(95%CI:5.8-16.6 copies/reaction)with linearity over a 3-log 10-unit range.Total HIV DNA in CD4^(+)T cells was positively associated with integrated HIV DNA(r=0.76,P<0.0001).Meanwhile,both total HIV DNA and integrated HIV DNA in CD4^(+)T cells were inversely correlated with the ratio of CD4/CD8 but positively correlated with the CD8^(+)T-cell counts.Conclusions:This ddPCR assay can quantify total HIV DNA and integrated HIV DNA efficiently with robustness and sensitivity.It can be readily adapted for measuring HIV DNA with non-B clades,and it could be beneficial for testing in clinical trials.

RASSF1A methylation as a biomarker for detection of colorectal cancer and hepatocellular carcinoma

BACKGROUND Studies have validated the potential of methylated cell-free DNA as a biomarker in various tumors,and methylated DNA in plasma may be a potential biomarker for cancer.AIM To evaluate the diagnostic value of RASSF1A methylation in plasma for colorectal cancer(CRC)and hepatocellular carcinoma(HCC).METHODS A total of 92 CRC patients,67 colorectal polyp(CRP)patients,63 HCC patients,and 66 liver cirrhosis(LC)patients were enrolled.The plasma DNA was subjected to DNA extraction,double-strand DNA concentration determination,bisulfite conversion,purification,single-strand DNA concentration determination,and digital polymerase chain reaction(PCR)detection.The methylation rate was calculated.The diagnostic value was evaluated by the area under the curve(AUC).RESULTS The age and sex in the CRC and CRP groups and the HCC and LC groups were also matched.The DNA methylation rate of RASSF1A in plasma in the CRC group was 2.87±1.80,and that in the CRP group was 1.50±0.64.DNA methylation of RASSF1A in plasma showed a significant difference between the CRC and CRP groups.The AUC of RASSF1A methylation for discriminating the CRC and CRP groups was 0.82(0.76-0.88).The AUCs of T1,T2,T3 and T4 CRC and CRP were 0.83(0.72-0.95),0.87(0.78-0.95),0.86(0.77-0.95),and 0.75(0.64-0.85),respectively.The DNA methylation rate of RASSF1A in plasma in the HCC group was 4.45±2.93,and that in the LC group was 2.46±2.07.DNA methylation of RASSF1A in plasma for the HCC and LC groups showed a significant difference.The AUC of RASSF1A methylation for discriminating the HCC and LC groups was 0.70(0.60-0.79).The AUCs of T1,T2,T3 and T4 HCC and LC were 0.80(0.61,1.00),0.74(0.59-0.88),0.60(0.42-0.79),and 0.68(0.53-0.82),respectively.CONCLUSION RASSF1A methylation in plasma detected by digital PCR may be a potential biomarker for CRC and HCC.

Rhinocerebral mucormycosis caused by Rhizopus oryzae in a patient with acute myeloid leukemia: A case report

BACKGROUND Rhinocerebral mucormycosis(RCM)is a rare fatal fungal infection which is on the increase among immunocompromised hosts such as patients who have had hematological cancers,or have received immunosuppressive drugs,corticosteroids,or other T cell suppressing agents.CASE SUMMARY We report a case of RCM caused by Rhizopus oryzae,one of the most common opportunistic pathogens,in a patient suffering from a fourth relapse of acute myeloid leukemia.The patient developed RCM after he had received long-term antibiotic agents and corticosteroids.The pathogen was isolated three times from nasal secretions collected from the deep parts of the nasal cavity and was identified by morphology and internal transcribed spacer sequencing.Blood infection was excluded by droplet digital polymerase chain reaction and blood culture.The patient was empirically treated with caspofungin and voriconazole for several days while the lesions continued to progress.The patient was given amphotericin B in combination with caspofungin after RCM was suspected,and the lesions improved over the course of treatment,which lasted several days.However,the patient eventually died of the primary disease.CONCLUSION This case indicates that immunosuppressive drugs,including corticosteroids and antimetabolites in hematological tumor,do increase the risk of infections of this type.Early diagnosis,prompt and frequent surgical debridement,and treatment with amphotericin B without delay are all essential in combatting RCM.

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.

Measurement and Characteristics of the Integration of China’s Trade in Services into Digital Global Value Chain

In this paper,the characteristics of digital trade in services of China are measured and revealed from the perspective of digitalization of trade in services.A digital global value chain is constructed and comprehensively analyzed at the three levels of path decomposition,two-way digital connection and bilateral connection.The study finds that the digital added value of China’s services driven by domestic demand outweighs that driven by foreign demand,and most countries engage in digital trade in services with China through simple participation;the role of China’s services in the digital global value chain is shifting from“digital value input”to“digital value output”,but its relative position is still low;China’s dependence on the import of digital intermediate products from developed countries has been significantly reduced,and the country has become the main source of digital intermediate imports for most countries,acting as a“hub”in the digital global value chain.This study comprehensively evaluates the strategic positioning and paths of integrating China’s services into the digital global value chain in multiple dimensions under the unifi ed accounting framework.It provides the reference for further improving the statistics accounting framework of China’s digital trade and promoting the high-quality development of the digital economy.

Functional relationship between ammonia-oxidizing bacteria and ammonia-oxidizing archaea populations in the secondary treatment system of a full-scale municipal wastewater treatment plant

The abundance of ammonia-oxidizing bacteria and archaea and their amo A genes from the aerobic activated sludge tanks,recycled sludge and anaerobic digesters of a full-scale wastewater treatment plant(WWTP)was determined.Polymerase chain reaction and denaturing gradient gel electrophoresis were used to generate diversity profiles,which showed that each population had a consistent profile although the abundance of individual members varied.In the aerobic tanks,the ammonia-oxidizing bacterial(AOB)population was more than 350 times more abundant than the ammonia-oxidizing archaeal(AOA)population,however in the digesters,the AOA population was more than 10 times more abundant.Measuring the activity of the amo A gene expression of the two populations using RT-PCR also showed that the AOA amo A gene was more active in the digesters than in the activated sludge tanks.Using batch reactors and dd PCR,amo A activity could be measured and it was found that when the AOB amo A activity was inhibited in the anoxic reactors,the expression of the AOA amo A gene increased fourfold.This suggests that these two populations may have a cooperative relationship for the oxidation of ammonia.