Multiple myeloma and Chagas disease: qPCR as a marker for preemptive antiparasitic therapy: a case reports series and review

ABSTRACT Multiple myeloma (MM) associated with Chagas disease is rarely described. This disease and its therapy suppress T cell and macrophage functions and increase regulatory T cell function, allowing the increase of parasitemia and the risk of Chagas Disease Reactivation (CDR). We aimed to analyze the role of conventional (cPCR) and quantitative Polymerase Chain Reaction (qPCR) for prospective monitoring of T. cruzi parasitemia, searching for markers of preemptive antiparasitic therapy in MM patients with Chagas disease. Moreover, we investigated the incidence and management of hematological diseases and CDR both inside and outside the transplant setting in the MEDLINE database. We found 293 studies and included 31 of them. Around 1.9-2.0% of patients with Chagas disease were reported in patients undergoing Stem Cell Transplantation. One case of CDR was described in eight cases of MM and Chagas disease. We monitored nine MM and Chagas disease patients, seven under Autologous Stem Cell Transplantation (ASCT), during 44.56±32.10 months (mean±SD) using parasitological methods, cPCR, and qPCR. From these patients, three had parasitemia. In the first, up to 256 par Eq/mL were detected, starting from 28 months after ASCT. The second patient dropped out and died soon after the detection of 161.0 par Eq/mL. The third patient had a positive blood culture. Benznidazole induced fast negativity in two cases; followed by notably lower levels in one of them. Increased T. cruzi parasitemia was related to the severity of the underlying disease. We recommend parasitemia monitoring by qPCR for early introduction of preemptive antiparasitic therapy to avoid CDR.

Establishment and verification of a real-time quantitative PCR method for determination of exogenous antibody gene copy number in CHO cells / 中国生物制品学杂志

@#Objective To establish a real-time quantitative PCR method using SYBR GreenⅠto detect the copy numbers of light chain(LC)and heavy chain(HC)of exogenous antibody gene in CHO cells,and verify and preliminarily apply this method.Methods With the B2m(β2-microglobulin)expressed stably in CHO cells as the internal reference gene,suitable primers of LC,HC genes and internal reference gene were designed respectively,and the reaction system and program of the real-time quantitative PCR method were determined. The established method was verified for the specificity,linearity,precision and durability,and used to detect the copy numbers of LC and HC genes in the recombinant cell lines of working cell bank(WCB)and cells of different passages.Results The primers of exogenous genes and internal reference gene showed specific binding to the target fragments;The efficiency of primer amplification for the B2m gene,LC gene,and HC gene was 106. 7%,106. 3% and 99. 1%,respectively,and the correlation coefficients of the linear equations were all greater than 0. 99 with a good linear relationship;The relative standard deviations(RSDs)of precision verification were all less than 1%;Few cycles of freeze-thaw in a short period had little effect on the detection results. The copy numbers of LC and HC genes in different generations of recombinant cell lines detected by the established method showed no obvious changes.Conclusion A real-time quantitative PCR method for the determination of the copy number of exogenous genes in CHO cells was successfully established with good specificity,linearity,precision and durability,which provides a reference for detecting the copy number of exogenous genes expressed in other CHO cell lines

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.

Preparation of the first national reference standard for determination of HEK293 cell DNA content / 中国生物制品学杂志

@#Objective To prepare a national reference standard for the quantification of HEK293 cell DNA content,so as to provide a support for the determination of residual DNA in HEK293 cells in the industry.Methods HEK293 cell DNA prepared using Genomic-tip 500/G and genomic DNA purification reagents was used as source materials,and the purity and content were assessed using ultraviolet spectrophotometry and agarose gel electrophoresis.After dilution to approximately 100 ng/μL,the DNA was aliquoted at 160 μL/tube.Five different laboratories were organized for collaborative calibration by using ultraviolet spectrophotometry, and the stability and applicability were evaluated.Results The HEK293 cell DNA national reference standard exhibited A_(260)/A_(280) ratios between 1.8 and 2.0 and displayed a single band on electrophoresis,meeting the specified criteria.Collaborative calibration across five laboratories yielded 78 valid data points with an average content of 104.8 ng/μL,a relative standard deviation(RSD) of 4.2%.The 95% confidence interval for the mean was 103.8—105.8 ng/μL,and the 95% reference range for single measurements was 96.0—113.6 ng/μL.The average confidence limit rate was 1.0%,and the recommended storage condition was-80 ℃.Applicability studies were conducted using two different models of fluorescence quantitative PCR instruments.The reference standard exhibited good applicability within the range of 0.3—3 000 pg/reaction,with amplification efficiencies of 101% and 95%,and R~2 values of 0.999 2 and 0.999 5 for the standard curves,respectively.Conclusion This batch of HEK293 cell DNA national reference standard meets all required specifications and can be utilized as a national reference standard for fluorescence quantitative PCR detection,with a certified content of 104.8 ng/μL,assigned batch number 270039-202301.

Exploration of cross-cultivar group characteristics of a new cultivar of Prunus mume 'Zhizhang Guhong Chongcui' / 生物工程学报

'Zhizhang Guhong Chongcui' is a new cultivar of Prunus mume with cross-cultivar group characteristics. It has typical characteristics of cinnabar purple cultivar group and green calyx cultivar group. It has green calyx, white flower, and light purple xylem, but the mechanism remains unclear. In order to clarify the causes of its cross-cultivar group traits, the color phenotype, anthocyanin content and the expression levels of genes related to anthocyanin synthesis pathway of 'Zhizhang Guhong Chongcui', 'Yuxi Zhusha' and 'Yuxi Bian Lü'e' were determined. It was found that the red degree of petals, sepals and fresh xylem in branches was positively correlated with the total anthocyanin content. MYBɑ1, MYB1, and bHLH3 were the key transcription factor genes that affected the redness of the three cultivars of flowers and xylem. The transcription factors further promoted the high expression of structural genes F3'H, DFR, ANS and UFGT, thereby promoting the production of red traits. Combined with phenotype, anthocyanin content and qRT-PCR results, it was speculated that the white color of petals of 'Zhizhang Guhong Chongcui' were derived from the high expression of FLS, F3'5'H, LAR and ANR genes in other branches of cyanidin synthesis pathway, and the low expression of GST gene. The green color of sepals might be originated from the relatively low expression of F3'H, DFR and ANS genes. The red color of xylem might be derived from the high expression of ANS and UFGT genes. This study made a preliminary explanation for the characteristics of the cross-cultivar group of 'Zhizhang Guhong Chongcui', and provided a reference for molecular breeding of flower color and xylem color of Prunus mume.

Establishment and evaluation of a method for extracting exogenous short DNA fragments of Schistosoma japonicum from urine samples / 中国血吸虫病防治杂志

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.

Detecting the copy number of orfA gene to identify Brucella species and biovars by a real-time quantitative PCR assay / 中华地方病学杂志

Objective:To establish a real-time quantitative PCR assay to detect the copy number of IS711 transposase gene (orfA) in Brucella genome, and the assay is applied to identify the species and biovars of Brucella. Methods:To establish an orfA gene copy number detection system based on Taqman real-time quantitative PCR technique. Primers and probes of bcsp31 and orfA genes were designed, the contents of bcsp31 gene and orfA gene in the same strain with the same DNA concentration were simultaneously detected by real-time quantitative PCR assay, and cycle number (CT value) of the two genes were obtained. According to the differences of CT values of bcsp31 gene and orfA gene, the copy number of orfA gene in Brucella genome was calculated. At the same time, the DNA of Brucella 16M strain was double decreasing dilution to verify the stability of the detection system. Results:A real-time quantitative PCR assay was used to detect bcsp31 gene and orfA gene simultaneously, when the DNA concentration difference of 16M strain was 2 times, the mean difference of CT values measured was 1.00, 95% confidence interval was 0.95-1.05, standard deviation was 0.17, and coefficient of variation was 0.17. The orfA gene copy number of 30 Brucella strains was detected by this detection system. It was found that there were 6, 9, and 7 copy numbers in the biovars 1-3 of Brucella melitensis, respectively. The strain of Brucella suis biovar 2 had 10 copy numbers, which were different from those of the other 4 strains of biovars 1, 3-5. There were 37 copy numbers in Brucella ovis strain. The copy numbers were stable at 5-6 copies in 8 biovars (1-7, 9) of Brucella abortus strains. Conclusions:A real-time quantitative PCR assay for detection of orfA gene copy number in Brucella DNA has been established. This method could identify some Brucella species and biovars strains.

Molecular quantification for quality control of Jinqian Baihuashe in Jinlong Capsule / 药学学报

The quality control of Chinese patent medicines containing animal-derived crude drugs is relatively difficult, because the effective constituents of most animal-derived crude drugs remain unknown. Even if there are relevant methods, they are usually qualitative, and quantitative indicators are either lacking or have poor specificity. This paper has proposed to use molecular quantitative technology to control the quality of Chinese patent medicines containing animal-derived crude drugs. In this study, a molecular quantitative method based on fluorescence quantitative PCR was established for the determination of Jinqian Baihua She in Jinlong Capsule. The method has good specificity, sensitivity, and repeatability. There is a good linear relationship between the content of DNA fragments and the CT (cycle threshold) value. The content of the Bungarus multicinctus-specific fragment in Jinlong Capsule is 24.1-46.6 IU·mg-1. It is suggested that the content of the specific fragment of Jinqian Baihua She should not be less than 19.3 IU·mg-1 as one of the quality control criteria of Jinlong Capsule. The study can provide a reference for the quality control of Chinese patent medicines containing animal-derived crude drugs.

Development and validation of real-time fluorescence quantitative PCR method for murine virus / 中国生物制品学杂志

@#Objective To develop and validate the real-time fluorescent quantitative PCR(Q-PCR)method for the detection of 8 murine viruses. Methods The specificity,sensitivity and precision of the Q-PCR method were verified by four laboratories,and the virus simulated contamination test and blind sample detection were carried out simultaneously,of which the detection results were compared. The Q-PCR method was used to detect 26 batches of monoclonal antibody cell lines for SARS-CoV-2 vaccine production and 15 batches of other products of murine origin. Results The Q-PCR method used for detecting 8 kinds of murine viruses had no cross reaction with the same family and genus or other murine viruses. Except the sensitivity of laboratory 2 to ectromelia virus(EctV/Mouse Pox,MPV)was 2 × 10~2copies/μL,the sensitivity of laboratory 2 to other 7 viruses and 3 other laboratories to 8 murine viruses was 2 × 10~1copies/μL. Except the inter-assay CV of the copy number of mouse adenovirus(MAdV)detected by laboratory 3 was 37. 58%,the intra-assay and inter-assay CVs of the Ct and copy number of other 7 viruses detected by laboratory 3 and those of 8 viruses detected by other 3 laboratories were all less than 25%.The sensitivity of virus simulated contamination test met the parameter requirements. The coincidence rate of blind sample detection results by 4 laboratories was 100%. All the 26 batches of monoclonal antibody cell lines for SARS-CoV-2 vaccine production and 15 batches of other murine derived products were negative for 8 murine viruses. Conclusion Q-PCR method for murine virus has good specificity,sensitivity and precision,and can be used for the detection of murine derived biological products.

Analysis of pathogenic characteristics of enteroaggregative Escherichia coli in children with diarrhea in Shanghai from 2019 to 2021 / 上海预防医学

ObjectiveTo understand the presence of virulence genes， molecular typing characteristics， and antibiotic sensitivity of enteroaggregative Escherichia coli （EAEC） in children with diarrhea in Shanghai， so as to provide a scientific basis for EAEC monitoring and standardized treatment of EAEC infection. MethodsEAEC strains isolated from children （≤5 years old） with diarrhea in six districts of Shanghai were collected as the study subjects. EAEC virulence genes were detected by real-time fluorescence quantitative PCR， molecular typing was performed by pulsed-field gel electrophoresis （PFGE）， and drug susceptibility tests were conducted using the microbroth dilution method. χ2 test and two independent samples t-test were used to compare the differences in virulence genes and antibiotic resistance between suburban and urban EAEC strains. ResultsFrom 2019 to 2021， the overall detection rates of gene aggR， pic and astA of 59 EAEC were 30.5%， 50.8%， and 57.6%， respectively. There was no significant difference in the detection rates of virulence genes between suburban and urban EAEC strains （P>0.05）. PFGE analysis revealed that only two EAEC strains belonged to the same PFGE pattern and were collected from the same hospital， and the overall PFGE patterns were polymorphic. EAEC showed susceptibility to imipenem and colistin E， and the resistance rates to sulfamethoxazole （SXT）， ampicillin （AMP）， nalidixic acid （NAL）， and tetracycline （93.1%， 79.3%， 63.8%， and 58.6%， respectively） were higher than 50.0%. The antibiotic resistance rates of cefazolin （CFZ）， cefotaxime （CTX）， and ciprofloxacin （CIP） were significantly different between EAEC strains from suburban and urban areas （P<0.05）. A total of 47 strains exhibited multi-drug resistance， with the most common resistance spectrum being AMP-SXT-NAL. There was no statistically significant difference in the number of multidrug-resistant EAEC strains between suburban and urban areas （P>0.05）. ConclusionThe EAEC virulence gene assemblages in children with diarrhea in the six districts of Shanghai are diverse， and the molecular typing patterns are relatively scattered， indicating possible cross-infection of homologous strains. Multi-drug resistance in EAEC strains is relatively common， and there is a statistically significant difference in the resistance rates of CFZ， CTX and CIP between urban and suburban EAEC strains. Attention should be given to standardizing the use of clinical antibiotics to effectively control the dissemination of multidrug-resistant EAEC strains.

Development and verification of multiplex fluorescence quantitative PCR (Taqman probe) for detection of telomerase activity / 中国生物制品学杂志

@#Objective To develop and verify a multiplex fluorescence quantitative PCR(Taqman probe) method for the detection of telomerase activity.Methods Specific reverse transcription primers,two pairs of quantitative primers and probes were designed for the CDS sequence of telomerase catalytic subunit telomerase reverse transcriptase(TERT).After optimization of the reverse transcription primers(specific reverse transcription primers and random primers) and quantitative primers(two pairs of quantitative primer probes used alone or in combination) in the reaction system,with the primer probe of internal reference gene GAPDH,multiplex fluorescence quantitative PCR was performed in a single tube.In addition,telomerase positive standard and negative standard were prepared with 293T and MRC-5 cells respectively,and the stability and precision of the method were verified.The telomerase activity in 19 normal mesenchymal cell samples and 32 breast cancer cell samples were detected by the developed method.Results The optimum reaction system was as follows:using cDNA synthesized with specific reverse transcription primers as the template,2 pairs of quantitative primer probes of TERT gene were mixed with internal reference gene GAPDH primer probes for multiplex fluorescence quantitative PCR reaction in a single tube.After optimization,the sensitivity and TERT fluorescence signal quantity of the system were greatly improved,and the ΔRn was enhanced by 3 times.The amplification curve of positive standard TERT gene was normal,and the ΔCt between TERT gene and GAPDH gene remained stable.The amplification curve of GAPDH gene in negative standard was normal,while there was no amplification curve of TERT gene.There was a little difference in ΔCt between TERT and GAPDH genes in the positive standard frozen and thawed for 3 and 5 times repeatedly and the positive standard without freezing and thawing,and the CVs of precision in intra-and inter-groups were all less than 1%.Telomerase activity was negative in 19 normal mesenchymal cell samples and positive in 32 breast cancer cell samples,and significant difference in Ct value of TERT gene between them was observed(t=4.236,P <0.001).Conclusion The developed multiplex fluorescence quantitative PCR(Taqman probe) method for the detection of telomerase activity has good stability and precision,which is expected to be used in early diagnosis and gene therapy of tumors.

Establishment and preliminary application of dual fluorescent quantitative PCR for detection of RV and CAV2 / 公共卫生与预防医学

Objective To investigate the shedding of CAV2-ΔE3-CGS after immunization and the background of canine adenovirus (CAV) infection, and to establish a dual fluorescent quantitative PCR detection method for rabies virus (RV) and canine adenovirus type 2 (CAV2). Methods A dual fluorescent quantitative PCR detection method was established by designing specific primers and probes for E1 gene of CAV and G gene of RV for the detection of CAV2-ΔE3-CGS. Oral swabs, anal swabs and environmental samples of stray dogs from experimental animal farm and dog detention center were tested. Results The standard curves generated by this method were Y=-3.351 × logX + 44.895, R2 = .999 and Y=-3.413 × logX + 45.192, R2=0.996, respectively. The linear relationships were good, and the minimum detection limits were both 102 copies/μL. CAV2-ΔE3-CGS was not detected in experimental animal farm. CAV was detected in dog detention center, and the positive rates were 5.88% (5/85) in oral swabs, 8.24% (7/85) in anal swabs, and 4% (1/25) in environmental samples. Conclusion The dual fluorescent quantitative PCR method can be used for the detection of CAV2-ΔE3-CGS after immunization and the investigation of CAV infection. The present study has shown that no CAV2-ΔE3-CGS has been detected after immunization and CAV infection rate of stay dogs is low in Shanghai. CAV2-ΔE3-CGS oral immunization meets requirement and is applicable.

Selection and validation of reference genes for quantitative real-time PCR analysis in Paeonia veitchii / 中国中药杂志

Paeonia veitchii and P. lactiflora are both original plants of the famous Chinese medicinal drug Paeoniae Radix Rubra in the Chinese Pharmacopoeia. They have important medicinal value and great potential in the flower market. The selection of stable and reliable reference genes is a necessary prerequisite for molecular research on P. veitchii. In this study, two reference genes, Actin and GAPDH, were selected as candidate genes from the transcriptome data of P. veitchii. The expression levels of the two candidate genes in different tissues(phloem, xylem, stem, leaf, petiole, and ovary) and different growth stages(bud stage, flowering stage, and dormant stage) of P. veitchii were detected using real-time fluorescence quantitative technology(qRT-PCR). Then, the stability of the expression of the two reference genes was comprehensively analyzed using geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that the expression patterns of Actin and GAPDH were stable in different tissues and growth stages of P. veitchii. Furthermore, the expression levels of eight genes(Pv-TPS01, Pv-TPS02, Pv-CYP01, Pv-CYP02, Pv-CYP03, Pv-BAHD01, Pv-UGT01, and Pv-UGT02) in different tissues were further detected based on the transcriptome data of P. veitchii. The results showed that when Actin and GAPDH were used as reference genes, the expression trends of the eight genes in different tissues of P. veitchii were consistent, validating the reliability of Actin and GAPDH as reference genes for P. veitchii. In conclusion, this study finds that Actin and GAPDH can be used as reference genes for studying gene expression levels in different tissues and growth stages of P. veitchii.

Expression Analysis of miRNA Profiles in Apheresis Platelets during Storage / 中国实验血液学杂志

OBJECTIVE@#To study the expression profiles changes of miRNA in apheresis platelets after 1, 3 and 5 days of storage.@*METHODS@#The apheresis platelets were collected from 20 volunteer blood donors. After mixing fully, the platelets were stored in a shaker with (22±2) ℃ horizontal oscillation. The samples were taken on the 1st, 3rd and 5th day, and used to sequence for miRNAs by DNA nanoball (DNB) sequencing technology, which were named as C_1, C_3 and C_5, respectively. The expression level of platelets miRNA was standardized by transcripts per kilobase million (TPM) algorithm. MiRNAs with P-value < 0.001 and the expression difference of more than two times were considered as significant difference between two groups. The expression of miRNAs was verified by real-time fluorescence quantitative PCR (RT-qPCR).@*RESULTS@#By DNB sequencing, there were 688, 730, and 679 platelet miRNAs expressed in C_1, C_3 and C_5 group, respectively. Cluster analysis showed that the expression profile of miRNAs changed significantly. The expression level of the first 20 high abundance miRNAs was about 4/5 of the total amounts of expressed miRNAs in each group, which the top five miRNAs were miR-21-5p, miR-26a-5p, miR-199a-3p, miR-126-3p, and let-7f-5p. The correlation of high abundance platelet miRNAs among the three groups was high (R2=0.876, R2=0.979, R2=0.937, respectively) and the differences were not statistically significant (P>0.05). Compared with the differential expression of platelet miRNAs with more than 1 000 TPM in the C_3 and C_1 group, there were 6 differentially expressed miRNAs, including 3 up-regulated (miR-146a-5p, miR-379-5p, and miR-486-5p) and 3 down-regulated (miR-652-3p, miR-142-5p, and miR-7-5p). While in the C_5 and C_1 group, there were 4 differentially expressed miRNAs, including 2 up-regulated (miR-146a-5p and let-7b-5p) and 2 down-regulated (miR-30d-5p and miR-142-5p). Compared with the differentially expression of platelet miRNAs between 1-1 000 TPM in the C_3 and C_1 group, there were 133 differentially expressed miRNAs, in which 99 were up-regulated and 34 were down-regulated. While in the C_5 and C_1 group, there were 77 differentially expressed miRNAs, in which 31 were up-regulated and 46 were down-regulated. The six selected differentially expressed miRNAs verified by RT-qPCR were consistent with those of sequencing.@*CONCLUSION@#The expression profiles of platelets miRNAs change significantly among 1, 3, and 5 d of storage in vitro.

Etiological analysis of acute respiratory tract infection in Taizhou of Zhejiang Province from 2020 to 2021 / 上海预防医学

ObjectiveWe examined the principal respiratory pathogens in patients with acute respiratory tract infection in Taizhou， Zhejiang Province during 2020‒2021 to provide evidence for prevention， diagnosis and treatment of acute respiratory tract infection. MethodsFrom September 2020 to August 2021， a total of 2 831 cases with acute respiratory tract infection were collected from two influenza sentinel surveillance hospitals in Taizhou， which had then received the examination of 22 respiratory pathogens by multiple fluorescence quantitative PCR. ResultsThe total positive rate of respiratory pathogens in 2 831 samples was 14.13%， among which enterovirus （7.77%） and respiratory syncytial virus （1.59%） were the principal pathogens. Except enterovirus， there was no significant difference in the positive rate of pathogens detected by gender（P>0.05）. Moreover， there was significant difference in pathogens by age （P<0.05）， with the highest positive rate in 0‒4 years（35.21%）. There was also significant difference in pathogens by seasons （P<0.05）， with the highest positive rate in summer（20.54%）. ConclusionThe positive rate of acute respiratory tract infection decreases significantly， compared with that before the COVID-19 epidemic. The differences in the positive rate differ significantly by age and seasons. Comprehensive consideration of diverse factors before diagnosis and the utilization of multiple fluorescent quantitative PCR can quickly and effectively determine the pathogens in the early stage of infection. Our findings may provide certain support for the diagnosis and treatment of acute respiratory infections in the context of COVID-19 in Taizhou.

Significance of serum circRNA hsa_circ_0000437 in the auxiliary diagnosis and prognosis of gastric cancer / 中华检验医学杂志

Objective:To investigate the expression of hsa_circ_0000437 in the serum of patients with gastric cancer and its clinical value.Methods:The serum samples from 80 patients (57 males and 23 females) with pathologically confirmed gastric cancer (GC), 50 gastric benign disease (28 males and 22 females) and 80 healthy controls (46 males and 34 females) were collected from October 2018 to December 2020 in Affiliated Hospital of Nantong University.Serum samples from 35 of 80 gastric cancer patients after operation were collected. The expression of serum hsa_circ_0000437 was determined by real-time fluorescent quantitative PCR (RT-qPCR). Serum carcinoembryonic antigen (CEA), carbohydrate antigen 199 (CA 199) and carbohydrate antigen 724 (CA724) were determined by chemiluminescence method.Comparisons of serum hsa_circ_0000437 between groups were performed by Mann-Whitney U test.The correlation between serum expression of hsa_circ_0000437 in gastric cancer patients and its clinical pathological characteristics was performed by χ 2 test.Receiver operating characteristic (ROC) curve and the area under the curve of ROC (AUC) were used to evaluate their diagnosis efficiency. Kaplan-Meier survival curve analysis was used to analyze the relationship between the expression level of serum hsa_circ_0000437 and the prognosis of patients. Results:The relative expression of hsa_circ_0000437 in GC, gastric benign disease, healthy controls were 2.252 (1.235, 4.765), 1.598(1.139, 1.982) and 1.000 (0.818, 1.385) respectively.The relative expression of hsa_circ_0000437 in GC was significantly higher than that in gastric benign disease ( P<0.001) and healthy controls ( P<0.001). The difference between gastric benign disease and healthy controls was also statistically significant ( P<0.001).The differences of serum hsa_circ_0000437 expression in GC patients between T stage, N stage, and tumor differentiation were statistically significant. The AUC of hsa_circ_0000437, CEA, CA199 and CA724 in GC patients were 0.863, 0.619, 657 and 0.608 respectively compared with healthy controls. The AUC of above four-parameter panel was 0.892 and the sensitivity was up to 97.5% (78/80). Kaplan-Meier survival curve showed that the overall survival rate of patients with high serum hsa_circ_0000437 expression was significantly lower than that of patients with low expression ( P=0.008). Conclusion:Serum hsa_circ_0000437 could be a biomarker for the auxiliary diagnosis and prognosis of GC.

Application and development of molecular diagnostic techniques for detection infectious diseases / 中华检验医学杂志

With the development of the concept of precision medicine, under the background of the new coronavirus pneumonia epidemic, the clinical diagnosis and treatment of infectious diseases has received more and more attention. The experimental diagnosis technology with molecular biology as the core is used as important means for the clinical laboratory diagnosis of infectious diseases. This lcind of technology is paid special attention. In recent years, advances in nanomaterials, applied chemistry, photophysics, and biosensing technologies have also ushered in revolutionary and creative developments in molecular diagnostic technology. This article reviews the application and development of the latest molecular diagnostic technologies, such as next-generation quantitative PCR technology and gene sequencing technology, isothermal amplification technology, biochip and biosensor technology in the clinical diagnosis of infectious diseases.

Establishment and application of detection method for interferon stimulated genes expression in children / 中华检验医学杂志

Objective:To establish the detection method for the interferon stimulated genes(ISGs), calculate the cut-off value and test it in clinical practice.Methods:Patients with type I interferonopathies who were admitted to Peking Union Medical College Hospital from November 2017 to September 2021 were chosen as the disease group, and healthy children were included as the control group. A total of 18 children were in the disease group, including 8 males and 10 females, with a median age of 8.5 years for the first test. From them 25 blood specimens were collected. A total of 28 healthy children, aged 1 to 18 years, with a median age of 10.5 years, including 15 males and 13 females, were included in the control group. Blood samples of 34 controls and 18 interferonopathies patients were collected, then total RNA extraction and cDNA synthesis were performed. Real-time quantitative polymerase chain reaction assays were run in duplicate to measure the expression of six ISGs: interferon induced protein with tetratricopeptide repeats 1 (IFIT1), interferon α inducible protein 27 (IFI27), interferon induced protein 44 like (IFI44L), interferon stimulated genes 15 (ISG15), sialic acid binding Ig like lectin 1 (SIGLEC1), and radical S-adenosyl methionine domain containing 2 (RSAD2). The relative abundances of each target transcript was normalized to the expression level of β-Actin and OAZ. The median fold change of the six ISGs was used to create an interferon score (IS) for each individual. Samples with abnormal expressions were removed and the cDNA mix of the remaining samples was used as a calibrator to calculate the IS. We define an abnormal IS as being greater than+2 standard deviations above the mean of controls. Differences in IS between groups were compared using t-test or Mann-Whitney U-test. Results:The mean IS of controls was 1.046, standard 0.755, and the cut-off value was 2.556. A total of 25 samples from 18 interferonopathies patients were tested. The mean value was 27.010 with a 15/18 abnormality rate. Compared with the control group, IS in patients was significantly higher, t=4.247( P=0.000 1). The accuracy, precision, sensitivity, and specificity were 91.30% (42/46), 7.47%(0.084/1.124), 15/18, and 96.43% (27/28), respectively. Conclusion:This study provides a new and reliable method for clinical screening and dynamic monitoring of type Ⅰ interferonopathies by detecting ISGs expression and creating an IS.

Establishment and preliminary application of a joint detection method for transplantation-associated infection pathogens / 中国输血杂志

【Objective】 To investigate the effectiveness of multilink real-time fluorescence quantitative PCR (qPCR) in the detection of common pathogens in transplantation. 【Methods】 The primers of the qPCR detection system were designed for 24 common infectious pathogens after clinical transplantation, and the standard plasmids of each pathogen were used to verify the qPCR reaction.After the primer probe effect and concentration of each pathogen reaction system in this experiment was optimized, the sensitivity, correlation coefficient (R2) and amplification efficiency (E) of qPCR method were analyzed and confirmed.Twenty-two samples from patients, who underwent liver and kidney transplantation in transplant ICU of Sichuan Provincial People′s Hospital, were used to verify the application of the detection system.The total nucleic acid of 100 μL was extracted from each individual and divided into two aliquots, which were detected by multi-link qPCR reaction system and analyzed by high-throughput sequencing method (NGS). At the same time, samples (2 mL each) were taken from the transplanted patients for microbial culture.The results of the three detection methods were compared, and the NGS method was taken as the gold standard to analyze the positive detection rate of the multi-link qPCR method and its difference with the culture method and NGS. 【Results】 The lower limit of qPCR detection for 24 pathogens in the established qPCR detection system was 101cp/μL(R2>0.99), with the positive rate of pathogens at 59.1% (13/22), showing significant difference versus microbial culture (18.2%, 4/22)(P<0.05), but not versus NGS (63.6%, 14/22)(P>0.05). Percentage of pathogens detected was as follows: human herpetic virus type 6 (HHV-6) 30.8% (4/13), cytomegalovirus (HCMV) 23.1% (3/13), Epstein-Barr virus (EBV) 23.1% (3/13), human parvovirus B19 15.4% (2/13), Haemophilus influenzae (Hin) 15.4% (2/13), Enterococcus faecium (EFM) 15.4% (2/13), Clostridium difficile 15.4% (2/13), Escherichia coli 7.7% (1/13), Stenotrophomonas maltophilia (Sma) 7.7% (1/13), Klebsiella pneumoniae (Kpn) 7.7% (1/13), Enterococcus faecalis (Efa) 7.7% (1/13) and Streptococcus pneumoniae (Spn) 7.7% (1/13). The consistency rate of pathogens detected by the three methods was 32% (7/22), among which the consistency rate of multi-link qPCR with NGS method was 59% (13/22), and multi-link qPCR with microbial culture was 41% (9/22). 【Conclusion】 Compared with the microbial culture, the multi-link qPCR method demonstrated high sensitivity, accurate quantification, short time and low cost for the detection of common pathogens in clinical transplantation.Multi-link qPCR combined with NGS and microbial culture is helpful to quickly predict the pathogen infection status of patients after transplantation.

Validation and application of a blood screening method for SARS-CoV-2 nucleic acid / 中国输血杂志

【Objective】 To evaluate the performance and clinical application of a high-throughput nucleic acid blood screening detection system for SARS-CoV-2, so as to provide basis and technical means for its application in detection of plasma from recovered COVID-19 patients. 【Methods】 The SARS-CoV-2 nucleic acid was detected by real-time fluorescence quantitative PCR, and the sensitivity, precision, anti-interference and other parameters were evaluated. Blood donor samples collected during COVID-19 epidemic period were screened using the detection system to evaluate its applicability. 【Results】 The detection limits of gene N and ORF 1ab were 3.98 copies/mL and 9.38 copies/mL, respectively. The CV of high and low concentration samples were both less than 5%. Hemoglobin at 500 mg/dL and triglyceride at 3g/dL had little effect on the results. The detection system can effectively prevent carryover, thus avoiding false positive results. The SARS-CoV-2 nucleic acid blood screening was carried out in a total of 39 306 blood samples, and all samples were negative. 【Conclusion】 The established method can meet the needs of SARS-CoV-2 nucleic acid screening therefore ensure the safe application of plasma from recovered COVID-19 patients.