| ObjectsThe aim of this study is to analysis the application and accuracy of the diagnostic tests applied in China in recent 10 years. The study is also to compare the efficiency of three DNA extraction methods and to establish a method of detecting Brucella by fluorescence quantitative PCR and PCR.MethodsA search was conducted in Chinese Journal of Zoonoses, Chineses Journal of Epidemiology, Chinese Journal of Endemiology, Chinese Journal of Control of Endemic Diseases and Disease Surveillance for papers about the diagnostic tests of human brucellosis in China published from 2004-2014. The data about the application and the validity of the tests was extracted from the included papers, then the data was merged and analyzed. In the DNA extraction assay, several variables (listed below) were controlled and compared to find out the feature and the optimized operating condition of the three methods. The extraction methods: the method of centrifugation column (TIANamp Bacteria DNA Kit), magnetic beads (Taco Total DNA Extraction Kit) and boiling in water. The concentration of Brucella melitansis 16m bacteria suspension:1011,1010.5,1010,109.5,109 CFU/ml. The elution volume:50 ?l,100 ?l, 200 ?l (except the boiling method, which do not have the elution step). The concentration and purity of the extracted nucleic acids was measured with Nanodrop ND-1000 spectrophotometer. The integrity of the nucleic acids was observed by agarose gel electrophoresis. The influence of each method on the downstream experiment was tested by PCR and fluorescence quantitative PCR. To develop the method of the estimation reference of the concentration of bacteria suspension through fluorescence quantitative PCR and PCR. The concentration of Brucella bacteria suspension was measured by plate counting method for more accurate count of live bacteria, the influence of different sample (bacteria suspended in normal saline and serum) and primer on the detection limits were compared for more sensitive measurement.Results1.157 papers, including 22 papers about the accuracy of diagnosis test, were included. The papers were reported by 24 provinces, containing up to 716 280 cases of samples, tested from 1954-2012 with 14 kinds of different diagnostic tests.2. Among the diagnostic tests, SAT was applied for 256 050 cases, in all 24 provinces; RBT, 224 699 cases, in 20 provinces; dermal sensitivity test,184 787 cases, in 10 provinces, etc.3. The sample size detected in each province was 114 031 in Henan, then,104 287 in Inner Mongolia,93 484 in Zhejiang, respectively. The number of tests applied in each province was 9 in Zhejiang,7 in Inner Mongolia,5 in Shandong and Heilongjiang, respectively.4. Controlled with SAT, the agreement rate of GICA and DIGFA was 99.77%(9 811/9 834); then PAT,97.73%(86/88); DAgS-ELISA,96.49%(110/114); RBT,94.55% (2465/2607); IgG ELISA,86.38%(2068/2394); IgM ELISA,76.23%(1825/2 394); CFT,69.17%(83/120); dermal sensitivity test,56.36%(31/55).5. The yield of the boiling method was far more than the other two methods. The centrifugation column was somewhat higher than magnetic beads. The yield of the bacteria suspension of 1011?1010.5 CFU/ml was highest by centrifugation column and magnetic beads; the concentration of the product of boiling was increased linearly with the increase of the concentration of bacteria suspension. The influence of elution volumn of centrifugation column method on yield was not significant. The best elution volumn of magnetic beads was 50 ?l.6. In the three methods, the purity of boiling was highest, centrifugation column was less, magnetic beads was the last.7. The three methods were all repeatable, stable, and feasible to downstream experiment.8. Taking bcsp31 as target gene, the typical amplification could be obtained in the range of 108?103CFU/ml. Standard curve:y=-3.0574x+46.171, R2=0.9968. Ct value was linear in the range of 108?103CFU/ml.9. The detection limit of two different sample (bacteria suspended in normal saline and serum) was in the same magnitude,103 CFU/ml for fluorescence quantitative PCR and 106 CFU/ml for PCR. And there was no significant difference in the Ct value of each concentration.10. Detecting the bacteria suspension with different primer by traditional PCR, IS313/IS639 was the most sensitive, its least titer that can detected was 105 CFU/ml, B4/B5 was 106 CFU/ml, brucl/bruc5 was 10'CFU/ml.Conclusions1. SAT was the mostly applied tests in China. Then was RBT. RBT was of high sensitivity and specificity.2. dermal sensitivity test and PAT were applied in only a few provinces in recent 10 years.3. CFT and Coomb's were less applied in only one or two provinces.4. GICA and DIGFA were of good sensitivity and specificity and easy to operate, that can be applied conveniently in the field. The sample size was not very large and was concentrated in Zhejiang province.5. The sample size of ELISA was also not very large and was concentrated in Inner Mongonia. Some sample were also tested in Shandong and Inner Mongolia. The sensitivity and specificity of ELISA were both well, and it was very important as it can detect IgG and IgM separately, indicating its value in identifying the course of brucellosis.6. Because of its simplicity, repeatability and the good quality of its product, the magnetic beads can be applied when the scale of the sample was large or followed by long chain PCR.7. The boiling method was cheap, fast, repeatable and effective, however, its product was almost totally cracked into small fragment. It can be used to large-scale, low-cost extraction that only followed with small fragment PCR.8. The logarithmic value of the concentration of bacteria suspension and the Ct value meet the formula y=-3.0574x+46.171. that can be used to calculate the content of Brucella.9. The detection limit of fluorescence quantitative PCR was 103 CFU/ml,1000 times lower than PCR.10. The least titer of IS313/IS639 that can detected was 105 CFU/ml, B4/B5 was 106 CFU/ml, brucl/bruc5 was 107 CFU/ml. |
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