Genetic mutation analysis of 22 patients with congenital absence of vas deferens: a single-center study†.

Congenital absence of the vas deferens (CAVD), a congenital malformation of the male reproductive system, causes obstructive azoospermia and male infertility. Currently, the cystic fibrosis transmembrane conductance regulator (CFTR) has been recognized as the main pathogenic gene in CAVD, with some other genes, such as adhesion G-protein-coupled receptor G2 (ADGRG2), solute carrier family 9 isoform 3 (SLC9A3), sodium channel epithelial 1 subunit beta (SCNN1B), and carbonic anhydrase 12 (CA12), being candidate genes in the pathogenesis of CAVD. However, the frequency and spectrum of these mutations, as well as the pathogenic mechanisms of CAVD, have not been fully investigated. Here, we sequenced all genes with potentially pathogenic mutations using next-generation sequencing and verified all identified variants by Sanger sequencing. Further bioinformatic analysis was performed to predict the pathogenicity of mutations. We described the distribution of the p.V470M, poly-T, and TG-repeat CFTR polymorphisms and identified novel missense mutations in the CFTR and SLC9A3 genes, respectively. Taken together, we identified mutations in the CFTR, ADGRG2, SLC9A3, SCNN1B, and CA12 genes in 22 patients with CAVD, thus broadening the genetic spectrum of Chinese patients with CAVD.

Spatial learning and memory deficits induced by prenatal glucocorticoid exposure depend on hippocampal CRHR1 and CXCL5 signaling in rats.

BACKGROUND: Prenatal synthetic glucocorticoid (sGC) exposure increases the susceptibility to cognitive and affective disorders in postnatal life. We previously demonstrated that prenatal sGC exposure results in an increase in corticotropin-releasing hormone (CRH) receptor type 1 (CRHR1) expression in the hippocampus of rats, and CRHR1 is involved in synapse formation via regulation of C-X-C chemokine ligand 5 (CXCL5) in hippocampus. We sought to investigate that the roles of CRHR1 and CXCL5 in learning and memory impairment caused by prenatal sGC exposure. METHODS: Pregnant rats were administered with saline or dexamethasone (DEX) from gestational day (GD) 14 to GD21. DEX offspring at 2-day old were treated with saline and CRHR1 antagonists (antalarmin and CP154526) for 7 days. Some DEX offspring received intra-hippocampal injection of AAV9 carrying CXCL5 gene. Spatial learning and memory was assessed by Morris water maze test. Immunofluorescence analysis was applied to show synapsin I and PSD95 signals in hippocampus. Synapsin I and PSD95 protein level and CXCL5 concentration were determined by western blotting and ELISA, respectively. Organotypic hippocampal slice cultures were used to investigate the effect of DEX on CXCL5 production in vitro. RESULTS: Both male and female DEX offspring displayed impairment of spatial learning and memory in adulthood. Synapsin I and PSD95 signals and CXCL5 levels were decreased in DEX offspring. DEX offspring with antalarmin and CP154526 treatment showed improved spatial learning and memory. Antalarmin and CP154526 treatment increased synapsin I and PSD95 signals and CXCL5 concentration in hippocampus. Bilaterally hippocampal injection of AAV9 carrying CXCL5 gene improved the spatial learning and memory and increased CXCL5 concentration and synapsin I and PSD95 levels in hippocampus. DEX dose-dependently suppressed CXCL5 production in cultured hippocammpal slices, which was prevented by antalarmin treatment. CONCLUSION: CRHR1 and CXCL5 signaling in the hippocampus are involved in spatial learning and memory deficits caused by prenatal DEX exposure. CRHR1 activation contributes to decreased CXCL5 production in hippocampus induced by prenatal DEX treatment. Our study provides a molecular basis of prenatal GC exposure programming spatial learning and memory.

Further identification of a 140bp sequence from amid intron 9 of human FMR1 gene as a new exon.

BACKGROUND: The disease gene of fragile X syndrome, FMR1 gene, encodes fragile X mental retardation protein (FMRP). The alternative splicing (AS) of FMR1 can affect the structure and function of FMRP. However, the biological functions of alternatively spliced isoforms remain elusive. In a previous study, we identified a new 140bp exon from the intron 9 of human FMR1 gene. In this study, we further examined the biological functions of this new exon and its underlying signaling pathways. RESULTS: qRT-PCR results showed that this novel exon is commonly expressed in the peripheral blood of normal individuals. Comparative genomics showed that sequences paralogous to the 140 bp sequence only exist in the genomes of primates. To explore the biological functions of the new transcript, we constructed recombinant eukaryotic expression vectors and lentiviral overexpression vectors. Results showed that the spliced transcript encoded a truncated protein which was expressed mainly in the cell nucleus. Additionally, several genes, including the BEX1 gene involved in mGluR-LTP or mGluR-LTD signaling pathways were significantly influenced when the truncated FMRP was overexpressed. CONCLUSIONS: our work identified a new exon from amid intron 9 of human FMR1 gene with wide expression in normal healthy individuals, which emphasizes the notion that the AS of FMR1 gene is complex and may in a large part account for the multiple functions of FMRP.

CRH/CRHR1 mediates prenatal synthetic glucocorticoid programming of depression-like behavior across 2 generations.

Pregnant women at risk of preterm labor usually receive synthetic glucocorticoids (sGCs) to promote fetal lung development. Emerging evidence indicates that antenatal sGC increases the risk of affective disorders in offspring. Data from animal studies show that such disorders can be transmitted to the second generation. However, the molecular mechanisms underlying the intergenerational effects of prenatal sGC remain largely unknown. Here we show that prenatal dexamethasone (Dex) administration in late pregnancy induced depression-like behavior in first-generation (F1) offspring, which could be transmitted to second-generation (F2) offspring with maternal dependence. Moreover, corticotropin-releasing hormone (CRH) and CRH receptor type 1 (CRHR1) expression in the hippocampus was increased in F1 Dex offspring and F2 offspring from F1 Dex female rats. Administration of a CRHR1 antagonist to newborn F1 Dex offspring alleviated depression-like behavior in these rats at adult. Furthermore, we demonstrated that increased CRHR1 expression in F1 and F2 offspring was associated with hypomethylation of CpG islands in Crhr1 promoter. Our results revealed that prenatal sGC exposure could program Crh and Crhr1 gene expression in hippocampus across 2 generations, thereby leading to depression-like behavior. Our study indicates that prenatal sGC can cause epigenetic instability, which increases the risk of disease development in the offspring's later life.-Xu, Y.-J., Sheng, H., Wu, T.-W., Bao, Q.-Y., Zheng, Y., Zhang, Y.-M., Gong, Y.-X., Lu, J.-Q., You, Z.-D., Xia, Y., Ni, X. CRH/CRHR1 mediates prenatal synthetic glucocorticoid programming of depression-like behavior across 2 generations.

Discovery of 2-((3-cyanopyridin-2-yl)thio)acetamides as human lactate dehydrogenase A inhibitors to reduce the growth of MG-63 osteosarcoma cells: Virtual screening and biological validation.

Lactate dehydrogenase A (LDHA) has emerged as an attractive target in the oncology field. In this paper, we present the identification of 2-((3-cyanopyridin-2-yl)thio)acetamide-containing compounds as LDHA inhibitors. The in vitro enzymatic assay suggested that inhibitor 9 had good inhibitory potency against LDHA with IC50 value as 1.24µM. Cytotoxicity assay showed that inhibitor 9 strongly inhibited the proliferation of cancer cell MG-63 (EC50=0.98µM). These findings indicated that inhibitor 9 could be employed as a lead for developing more potent LDHA inhibitor with anti-proliferative potency.

meso-Tetrakis[4-(heptyloxy)phenyl]porphyrin.

The core of the novel title centrosymmetric porphyrin derivative, C72H86N4O4, with long flexible hexyloxy substituents, is almost planar, which is anticipated to facilitate π-electron delocalization and lead to a significant deviation between the planes of the benzene rings and the molecular plane. The two N-bound H atoms on the pyrrole rings are disordered and the occupancy factors refined to a ratio of 0.28 (2):0.72 (2).

Proteomics analyses of acute kidney injury biomarkers in a rat exertional heat stroke model.

The frequency of exertional heat stroke (EHS) increases with the gradual elevation of global temperatures during summer. Acute kidney injury (AKI) is a common complication of EHS, and its occurrence often indicates the worsening of a patient's condition or a poor prognosis. In this study, a rat model of AKI caused by EHS was established, and the reliability of the model was evaluated by HE staining and biochemical assays. The expression of kidney tissue proteins in the EHS rats was analyzed using label-free liquid chromatography-tandem mass spectrometry. A total of 3,129 differentially expressed proteins (DEPs) were obtained, and 10 key proteins were finally identified, which included three upregulated proteins (Ahsg, Bpgm, and Litaf) and seven downregulated proteins (medium-chain acyl-CoA synthetase 2 (Acsm2), Hadha, Keg1, Sh3glb1, Eif3d, Ambp, and Ddah2). The qPCR technique was used to validate these 10 potential biomarkers in rat kidney and urine. In addition, Acsm2 and Ahsg were double-validated by Western blotting. Overall, this study identified 10 reliable biomarkers that may provide potential targets for the treatment of AKI caused by EHS.

Clinical Analysis of the Discovery of Malignant Gynecological Tumors in the Diagnosis and Treatment of Pelvic Organ Prolapse.

Background: Clinically, malignant gynecological tumors found by chance during the diagnosis and treatment of pelvic organ prolapse (POP) are rare, and they are usually missed, leading to delayed diagnosis and treatment. The initial treatment of these tumors cannot be standardized, and, as a single surgical intervention may not be able to treat both the tumor and prolapse, secondary surgery is usually needed, affecting the quality of life of patients. Case presentation: The present study retrospectively analyzed the data of three patients who were diagnosed with malignant gynecological tumors during the diagnosis and treatment of POP. These patients were among 215 patients with POP treated in Yuncheng Central Hospital of Shanxi Province between January 2011 and May 2020. The case characteristics, surgical interventions, postoperative treatments, and follow-ups were summarized, and the characteristics of diagnosis and treatment were analyzed in the context of relevant literature. Conclusion: As long as clinicians operate in strict accordance with the standards of diagnosis and treatment, obtain a complete medical history, undertake a physical examination, and remain diligent in auxiliary examinations, following existing clinical methods and diagnosis and treatment processes, patients with POP complicated with malignant gynecological tumors can be clearly diagnosed before and during surgery. In this way, initial treatment can be standardized, and surgical methods can be selected that address both the tumor and prolapse, thereby avoiding secondary surgery and improving the patient's quality of life.

Oxidative Damage to BV2 Cells by Trichloroacetic Acid: Protective Role of Boron via the p53 Pathway.

This study aimed to investigate the neurotoxicity induced by trichloroacetic acid (TCA) and the possible protective mechanisms of boron (B). Mouse BV2 cells were treated with TCA (0, 0.39, 0.78, 1.56, 3.12, 6.25, or 12.5 mmol/L) and B (0, 7.8, 15.6, 31.25, 62.5, 125, 500, or 1,000 mmol/L) for 3 h and 24 h, respectively. Then, reactive oxygen species, and supernatant proinflammatory cytokine and protein levels were analyzed after 24 h of combined exposure. Beyond the dose-dependent decrease in the cellular viability, it clearly increased after B supplementation ( P < 0.05). Moreover, B decreased oxidative damage, and significantly down-regulated IL-6 levels and up-regulated TNF-ß production ( P < 0.05). B also decreased apoptosis via the p53 pathway. The present findings indicated that TCA may induce oxidative damage, whereas B mitigates these adverse effects by decreasing cell apoptosis.

Verification on the Developmental Toxicity of Short-term Exposure to Phenol in Rats.

OBJECTIVE: To verify the health advisory for short-term exposure to phenol. METHODS: The method of this validation experiment was the same as the US Environmental Protection Agency (EPA) methodology for toxicology experiments used to determine phenol drinking water equivalent level (DWEL). Pregnant female Sprague-Dawley rats were administered phenol in distilled water by gavage at daily doses of 15, 30, 60, 120, and 240 mg/kg body weight (b.w.) from implantation (the 6th day post-mating) to the day prior to the scheduled caesarean section (the 20th day of pregnancy). The following information was recorded: general behavior; body weight; number of corpus luteum, live birth, fetus, stillbirth, and implantation; fetal gender; body weight; body length; tail length; and abnormalities and pathomorphological changes in the dams. RESULTS: In the 60 mg/kg b.w. dose group, the mortality of pregnant rats increased with increasing doses, suggesting maternal toxicity. Fetal and placental weights decreased as phenol dose increased from 30 mg/kg b.w., and were significantly different compared those in the vehicle control group, which suggested developmental toxicity in the fetuses. However, the phenol-exposed groups showed no significant change in other parameters compared with the vehicle control group ( P > 0.05). CONCLUSION: Despite using the same method as the US EPA, a different NOEAL of 15 mg/(kg·d) was obtained in this study.

Splicing of exon 9a in FMR1 transcripts results in a truncated FMRP with altered subcellular distribution.

FMRP is an RNA-binding protein, loss of which causes fragile X syndrome (FXS). FMRP has several isoforms resulted from alternative splicing (AS) of fragile X mental retardation 1 (FMR1) gene, but their biological functions are still poorly understood. In the analysis of alternatively spliced FMR1 transcripts in the blood cells from a patient with FXS-like phenotypes (normal CGG repeats and no mutation in coding sequence of FMR1), we identified three novel FMR1 transcripts that include a previously unidentified microexon (46 bp), terming the exon 9a. This microexon exists widely in unaffected individuals, inclusion of which introduces an in-frame termination codon. To address whether these exon 9a-containing transcripts could produce protein by evading nonsense-mediated decay (NMD), Western blot was used to analysis blood cell lysate from unaffected individuals and a 34 kDa protein that consistent in size with the molecular weight of the predicted truncated protein produced from mRNA with this microexon was found. Meanwhile, treatment of peripheral blood mononuclear cells with an inhibitor of NMD (Cycloheximide) did not result in significant increase in exon 9a-containing transcripts. Using confocal immunofluorescence, we found the truncated protein displayed both nuclear and cytoplasmic localization in HEK293T and HeLa cells due to lacking C-terminal domains including KH2, NES, and RGG, while the full-length FMRP protein mainly localized in the cytoplasm. Therefore, we hypothesize that the inclusion of this microexon to generate exon 9a-containing transcripts may regulate the normal functionality of FMRP, and the dysregulation of normal FMRP due to increased exon 9a-containing alternatively spliced transcripts in that patient may be associated with the manifestation of FXS phenotype.

Poly[diaqua-(µ-oxalato)(µ-2-oxido-pyridinium-3-carboxyl-ato)praseo-dymium(III)].

In the title complex, [Pr(C(6)H(4)NO(3))(C(2)O(4))(H(2)O)(2)](n), each Pr(III) ion is coordinated by eight O atoms from two 2-oxynicotinate ligands, two oxalate ligands and two water mol-ecules, displaying a distorted bicapped square-anti-prismatic geometry. The carboxyl-ate groups link adjacent praseodymium metal centres, forming layers parallel to the bc plane. The crystal packing is stabilized by inter-molecular O-H⋯O and N-H⋯O hydrogen bonds.

3-Hydr-oxy-1,2-dimethoxy-anthraquinone.

The title compound, C(16)H(12)O(5), was isolated from Morinda officinalis How. The anthraquinone ring system is almost planar, the dihedral angle between the two benzene rings being 1.12 (4)°. In the crystal structure, O-H⋯O and C-H⋯O hydrogen bonds link the mol-eculesin the crystallographic a-axis direction. Weak π-π stacking inter-actions [centroid-centroid distance between symmetry-related benzene rings of 3.699 (4) Å] are also present.

[Effects of progesterone on intracellular free Ca2+ concentration in the spermatozoa of fertile men and patients with unexplained infertility].

OBJECTIVE: To investigate the difference in the responsiveness of intracellular free Ca2+ concentration ([Ca2+]i) to progesterone in the spermatozoa of normal fertile men and patients with unexplained infertility. METHODS: Nine normal fertile men and 10 patients with unexplained infertility were selected in this study. After swim-up separation of the motile fraction and 2-hour in vitro capacitation, the spermatozoa were loaded with the fluorescent calcium indicator Fluo-3/AM (8.85 micromol/L) for 40 minutes away from the light, and then the sperm suspension was mixed with equal amount of 20% gelatin to immobilize the spermatozoa. The basal intracellular free [Ca2+]i and that induced by 10 micromol/L progesterone in the individual sperm were assessed by laser scanning confocal microscopy. RESULTS: The infertile patients had a significantly lower basal level of [Ca2+]i in the capacitated sperm than the fertile men (P < 0.01). The sperm from the normal controls responded to progesterone by exhibiting a rapid but transient rise in [Ca2+]i, with the peak level significantly higher than the basal level (P < 0.05), while those from the infertile patients by showing a slight increase, with no significant difference between the peak and basal levels (P > 0.05). Both the peak of the progesterone-induced [Ca2+]i and its increase amplitude expressed as the difference between the peak and basal levels were significantly higher in the normal fertile group than in the infertile patients (P < 0.01). CONCLUSION: The responsiveness of [Ca2+]i to progesterone is reduced in the spermatozoa of patients with unexplained infertility, which suggests a functional defect in the non-genomic sperm membrane progesterone receptor responsible for calcium influx.

Degradation of acenaphthene by ozone.

OBJECTIVE: To investigate the oxidation of acenaphthene (Ace), a polycyclic aromatic hydrocarbon (PAH) with a saturated C-C bond by ozone and to characterize the intermediate products of ozonation. METHODS: Ozone was generated from filtered dry oxygen by an ozone generator and continually bubbled into a reactor containing 1g/L Ace dissolved in an acetonitrile/water solvent mixture (90/10, v/v) at a rate of 0.5 mg/s. HPLC was used to analyze the Ace concentration. Total organic carbon (TOC) was used to measure the amount of water soluble organic compounds. GC-MS was used to identify the ozonized products. Oxygen uptake rate (OUR) of activated sludge was used to characterize the biodegradability of ozonized products. RESULTS: During the ozonation process, Ace was degraded, new organic compounds were produced and these intermediate products were difficult mineralize by ozone, with increasing TOC of soluble organics. The ozonized products were degraded by activated sludge more easily than Ace. CONCLUSION: Ozonation decomposes the Ace and improves its biodegradability. The ozonation combined with biological treatment is probably an efficient and economical way to mineralize acenaphthene in wastewater.

[Radioligand binding assay of progesterone receptors on normal fertile human sperm membrane].

OBJECTIVE: To investigate the progesterone-binding site on the normal fertile human sperm membrane after 2 hours of in vitro capacitation. METHODS: Viable spermatozoa were selected by a swim-up method. After 2 hours of in vitro capacitation, multipoint saturation binding experiments were performed. Sperm suspension and increasing concentrations of progesterone-11alpha-glucuronide-[125I] iodotyramine (125I-P) were added to 7 total binding tubes respectively, and equal amounts of sperm suspension and 125I-P were added to another 7 corresponding non-specific binding tubes in the presence of 10 micromol/L progesterone. After incubation for 1 hour at 4 degrees C, the radioactivity of both the tubes and the pellets after centrifugation was measured respectively. The equilibrium dissociation constant (Kd) and maximum binding capacity (Bmax) were calculated using the mathematical model of single site multi-point saturation method of Scatchard function and least-squares regression. RESULTS: Kd was (0.61 +/- 0.04) nmol/L and Bmax was (830 +/- 344) sites/cell. The significance test of the regression equation indicated that r = -0.980, P < 0.01. CONCLUSION: There is a high affinity and low capacity binding site for the progesterone (progesterone receptor) on the normal fertile human sperm membrane.

[Study on detection method of 8-OH-dG in DNA extracted from HepG2 cells in vitro by capillary zone electrophoresis].

OBJECTIVE: To establish an optimized method to detect 8-OH-dG after DNA oxidation damage by capillary zone electrophoresis. METHODS: HepG2 cell was used as target cell and conditions for the separation and detection were obtained by studying the influence of pH of the running buffer, temperature, running voltage on the separation. 0 and 15 mmol/L H2O2 were added into two groups of HepG2 cells (5 x 10(7)) respectively for 24h. DNA was extracted by saturated salting out method to avoid the formation of additional 8-OH-dG by the method of phenol/chloroform extraction. DNA samples were digested to free nucleotides by incubation overnight at 37 degrees C with a mixture of DNase I, snake venom phosphoatase and alkaline phosphate. Proteins were removed and the supernatant was neutralized and then extracted with diethyl ether. The residue was evaporated to dryness and reconstituted and then analyzed under the optimized conditions by capillary zone electrophoresis. RESULTS: The optimized conditions were: uncoated silica capillary (47 cm x 50 microm i.d.), 20 mmol/L borate buffer (pH 9.5), 25 degrees C, 25kV. The sample was injected by hydrostatic method for 20s. In either of H2O2-treated group and H2O2-untreated group, the peak of 8-OH-dG was detected. The 8-OH-dG content of H2O2-untreated DNA increased. CONCLUSION: The method is convenient, rapid, sensitive and cheap and safe. It provides an experimental platform to the application of 8-OH-dG in the biological monitoring of population.

Characteristics and application of established luciferase hepatoma cell line that responds to dioxin-like chemicals.

AIM: To establish a luciferase reporter cell line that responds dioxin-like chemicals (DLCs) and on this basis to evaluate its characteristics and application in the determination of DLCs. METHODS: A recombinant luciferase reporter plasmid was constructed by inserting dioxin-responsive element (DREs) and MMTV promoter segments into the pGL(3)-promoter plasmid immediately upstream of the luciferase gene, which was structurally demonstrated by fragment mapping analysis in gel electrophoresis and transfected into the human hepatoma cell line HepG(2), both transiently and stably, to identify the inducible expression of luciferase by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). The time course, responsive period, sensitivity, structure-inducibility and dose-effect relationships of inducible luciferase expression to DLCs was dynamically observed in HepG(2) cells stably transfected by the recombinant vector (HepG(2)-Luc) and compared with that assayed by ethoxyresorufin-O-deethylase (EROD) in non-transfected HepG(2) cells (HepG(2)-wt). RESULTS: The inducible luciferase expression of HepG(2)-Luc cells was noted in a time-, dose-, and AhR-dependent manner, which peaked at 4 h and then decreased to a stable level at 14 h after TCDD treatment. The responsiveness of HepG(2)-Luc cells to TCDD induction was decreased with culture time and became undetectable at 10th month of HepG(2)-Luc cell formation. The fact that luciferase activity induced by 3, 3', 4, 4'-PCB in HepG(2)-Luc cells was much less than that induced by TCDD suggests a structure-inducibility relationship existing among DLCs. Within the concentrations from 3.5 x 10(-12) to 5 x 10(-9) mol/L, significant correlations between TCDD doses and EROD activities were observed in both HepG(2)-luc and HepG(2)-wt cells. The correlation between TCDD doses from 1.1 x 10(-13) to 1 x 10(-8) mol/L and luciferase activities was also found to be significant in HepG(2)-luc cells (r=0.997, P<0.001), but not in their HepG(2)-wt counterparts. For the comparison of the enzyme responsiveness between cell lines to TCDD, the luciferase sensitivity and reproducibility in HepG(2)-luc cells were both better than that of EROD in HepG(2)-wt cells, the former was at 1.1 x 10(-13) mol/L and 3.5 x 10(-12) mol/L, and the coefficients of variation (CV) of the latter was 15-30 % and 22-38 %, respectively. CONCLUSION: The luciferase expression of HepG(2)-luc cells established in the present study could sensitively respond to the DLCs stimulation and might be a prospective tool for the determination of DLCs.

Improvement of chemically-activated luciferase gene expression bioassay for detection of dioxin-like chemicals.

OBJECTIVE: To improve the chemically-activated luciferase expression (CALUX) bioassay for detection of dioxin-like chemicals (DLCs) based on the toxicity mechanisms of DLCs. METHODS: A recombinant vector was constructed and used to transfect human hepatoma (HepG2). The expression of this vector was 10-100 folds higher than that of pGL2 used in previous experiments. The transfected cells showed aromatic hydrocarbon receptor (AhR)-meditated luciferase gene expression. The reliability of luciferase induction in this cell line as a reporter of AhR-mediated toxicity was evaluated, the optimal detection time was examined and a comparison was made by using the commonly used ethoxyresoufin-O-deethylase (EROD) activity induction assay. RESULTS: The results suggested that the luciferase activity in recombinant cells was peaked at about 4 h and then decreased to a stable activity by 14 h after TCDD treatment. The detection limit of this cell line was 0.11 pmol/L, or 10-fold lower than in previous studies, with a linear range from 1 to 100 pmol/L, related coefficient of 0.997, and the coefficient of variability (CV) of 15-30%. CONCLUSION: The luciferase induction is 30-fold more sensitive than EROD induction, the detection time is 68 h shorter and the detection procedure is also simpler.