Urinary Myeloperoxidase to Creatinine Ratio as a New Marker for Diagnosis of Urinary Tract Infection / 中国医学科学杂志(英文版)

Objective To determine whether urinary myeloperoxidase to creatinine ratio (MCR) can serve as a marker for diagnosis of urinary tract infection (UTI).Methods Patients suspected of UTI were consecutively enrolled and further divided into the culture positive and the sterile groups according to urine culture results. Subsequently, MCR, white blood cell (WBC) and bacteria in the urinary samples from patients were detected and compared between the two groups.Results Finally, 253 patients were enrolled including 157 urine culture positive patients and 96 urine culture negative patients (sterile group). After logarithmic transformation in 2 as the base, the MCR, WBC, and bacteria were separately presented as log, log(quantitative) , and log. The values of log(8.6±2.5 vs. 5.4±1.5, t=-12.453, P=0.001), log(quantitative) (8.0±2.5 vs. 5.2±1.8, t=-10.332, P=0.001), log (11.4±2.5 vs. 8.2±2.8, t=-9.297, P=0.001) and WBC (semi-quantitative) [2 (interquartile range 1, 3) vs. 1 (interquartile range 0.5, 1), Z=-7.580, P=0.001] showed significant difference between the urine culture positive group and the sterile group. Among the urine culture positive group, the values of log of the gram positive and gram negative subgroups were 7.2±2.5 and 9.0±2.4 (t=4.016, P=0.001), respectively. The correlation between log and log (quantitative), log, WBC (semi-quantitative) was 0.708 (Pearson correlation, P=0.001), 0.381 (Pearson correlation, P=0.001), and 0.606 (Spearman correlation, P=0.001), respectively. Conclusions MCR is positively correlated with WBC counts and could be served as a promising biomarker for diagnosis of UTI. MCR could be even used for initial inference of infectious bacteria types of UTI.

A novel modi cation of real-time AS-qPCR by using locked nucleic acid-modified oligonucleotide probe as wild type allele amplification blockers for quantitative detection of the JAK2 V617F mutation / 中华血液学杂志

<p><b>OBJECTIVE</b>To develop a novel real-time PCR for sensitively quantitative detection of JAK2 V617F allele burden in peripheral blood.</p><p><b>METHODS</b>Based on the real-time allele-specific PCR (AS-qPCR), the locked nucleic acid (LNA)-modified oligonucleotide probe was used for selectively blocking amplification of wild-type alleles in AS-qPCR, and then a novel AS-LNA-qPCR method was established. The percentages of sample JAK2 V617F alleles were directly calculated by its threshold cycle (Ct) values according to the standard curve which generated by JAK2 V617F alleles with its Ct values. We validated intra- and inter-assay variability for quantifying JAK2 V617F. We also assayed 623 apparent healthy donors by our method to validate its clinical application value.</p><p><b>RESULTS</b>The quantitative lower limit of this method for JAK2 V617F was 0.01%, and the intra- and inter-assay average variability for quantifying percentage of JAK2 V617F in total DNA was 6.3% and 8.6%, respectively. Nineteen JAK2 V617F-positive individuals were identified using AS-LNA-qPCR in blood of 623 apparently healthy donors, and the range of percentages of JAK2 V617F alleles were 0.01%-5.49%.</p><p><b>CONCLUSION</b>The AS-LNA-qPCR with highly sensitive and reproducible quantification of JAK2 V617F mutant burden can be used clinically for diagnosis as well as evaluation of disease prognosis and efficacy of therapy in patients with myeloproliferative neoplasms.</p>

Detection of JAK2V617F mutation rate by real-time fluorescent quantitative PCR using allele specific primer and TaqMan-MGB probe for dual inhibiting amplification of wild type alleles / 中国实验血液学杂志

This study was purposed to develop a real-time PCR assay for sensitive quantification of JAK2V617F allele burden in peripheral blood and to evaluate the clinical value of this method. Both allele-specific mutant reverse primer and wild-type TaqMan-MGB probe were used for dual-inhibiting amplification of wild-type alleles in a real-time PCR, and then the JAK2V617F mutant alleles were amplified specially. The standard curve for quantification of JAK2V617F was established by percentages of JAK2V617F alleles with threshold cycle (Ct) values in a real-time PCR. Furthermore, 89 apparent healthy donors were tested by this method. The results showed that the quantitative lower limit of this method for JAK2V617F was 0.1%, and the intra- and inter-assay average variability for quantifying percentage of JAK2V617F in total DNA was 4.1% and 6.1%, respectively. Two JAK2V617F-positive individuals were identified (the percentage of JAK2V617F alleles were 0.64% and 0.98%, respectively) using this method in blood from 89 apparently healthy donors. It is concluded that the developed method with highly sensitive and reproducible quantification of JAK2V617F mutant burden can be used clinically for diagnosis and evaluation of disease prognosis and efficacy of therapy in patients with myeloproliferative neoplasms. Moreover, this technique can be also used for quantitative detection of variety of single nucleotide mutation.

Cyclooxygenase inhibitors in some dietary vegetables inhibit platelet aggregation function induced by arachidonic acid / 中国实验血液学杂志

The study was purposed to investigate whether the cyclooxygenase inhibitors from some dietary vegetables can inhibit platelet aggregation function by the arachidonic acid (AA). The vegetable juice was mixed with platelet rich plasma (PRP), and asprin was used as positive control. The maximum ratio of platelet aggregation induced by AA was measured on the aggregometer; heme and cyclooxygenase-1 (COX(1)) or cyclooxygenase-2 (COX(2)) were added to test tubes containing COX reaction buffer, the mixture was vortex-mixed and exposed to aspirin or vegetable juice, followed by addition of AA and then hydrochloric acid (1 mol/L) was added to stop the COX reaction, followed by chemical reduction with stannous chloride solution. The concentration of COX inhibitors was detected by the enzyme immunoassay kit; vegetable juice (aspirin as positive control) was mixed with whole blood, which was followed by the addition of AA, and then the reaction was stopped by adding indomethacin, centrifuged, then the supernatant was collected, and the plasma thromboxane B(2) (TXB(2)) was measured by radioimmunoassay. The results showed that spinach juice, garlic bolt juice, blanched garlic leave juice and Chinese leek juice could inhibit by 80% human platelet aggregation induced by AA. 4 kinds of vegetables were all found a certain amount of cyclooxygenase inhibitors, which COX(1) and COX(2) inhibitor concentrations of spinach were higher than that of aspirin; 4 vegetable juice could significantly reduce the human plasma concentrations of TXB(2) induced by AA (p < 0.05). It is concluded that 4 kinds of raw vegetables containing cyclooxygenase inhibitors inhibit the production of TXA(2) and thus hinder platelet aggregation. Raw spinach, garlic bolt, blanched garlic and chinese leek inhibit significantly AA-induced human platelet aggregation in vitro. 4 kinds of vegetables may have a good potential perspective of anti-platelet aggregation therapy or prevention of thrombosis.

Mutation of cystathionine beta-synthase gene(CBS G919A/T833C)in patients with diabetic nephropathy detected by amplification refractory mutation system-real time PCR / 中华检验医学杂志

Objective To develop a TaqMan fluorogenic probe based amplification refractory mutation system (TaqMan-ARMS) for the detection of the C BS gene G919A and T833C mutations,and to investigate whether the mutations are associated with diabetic nephropathy in Chinese population. Methods According to the principle of amplification refractory mutation system,the cycle threshold (Ct) of wild (Wct) and mutation (Mct) allele-specific primers between the two PCR reactions in real-time PCR were monitored and genotype detection criteria were established based on the threshold ratio Act (?ct= Wct/ Mct) or an appearance of exponential amplification.With this technique,the G919A and T833C mutations in the CBS gene were analyzed in 94 patients with diabetic nephropathy and 140 control subjects. Results The detection criteria of TaqMan-ARMS assay for T833 allele were ?ct40,for T833C allele 0.940,respectively.The criteria for G919 allele were ?ct40,for G919A allele 0.9240,respectively.The T833C rates of TT,TC and CC genotypes were 98.94%, 1.06% and 0 in the patients and 99.29%,0.71% and 0 in the controls.The 833C allele frequencies were 0.53% in the patients and 0.36% in the controls.No significant differences in both genotypes and allele of T833C mutation were observed between the two groups.The G919A point mutation was not observed in all subjects.Conclusions A TaqMan-ARMS assay for the detection of the CBS gene GA919A and T833C mutations has been developed.The assay is accurate and sensitive,and is suitable for high-throughput detection of the point mutations.The G919A and T833C point mutations of CBS gene may not be related to diabetic nephropathy in Chinese.

Clinical and molecular biological features of 2 Chinese patients with Kennedy's disease diagnosed by gene analysis / 中华神经科杂志

Objective To diagnose Kennedy's disease (KD) via molecular analysis of the androgen receptor gene with suspected KD.Methods Two patients with suspected KD were reported.We analyzed their clinical features and investigated the number of CAG repeats in the androgen receptor genes. Results Both of the patients were characterized by slow progression of predominant proximal and bulbar muscle weakness.Patient 2 had oligospermatism.Serum creatine kinase and triglyceride levels were found markedly increased.The exact number of CAG was 52 in patient 1 and 48 in patient 2,respectively.These 2 patients were finally diagnosed as Kennedy's disease through the analysis of androgen receptor gene by PCR and direct sequencing.Conclusions The method of molecular analysis for KD had been copied in China.The clinical and molecular biological features of 2 Chinese patients with KD had been discussed.KD is a neurodegenerative disorder by proximal limb muscular atrophy and weakness with lower motor neuron signs,bulbar involvement.Dyscrinism and metabolic abnormalities may also be observed.Gene analysis is the unique and reliable methods to diagnose KD.