Method for detecting <i>Brucella </i>infection and application thereof

Abstract

The present invention provides a method for detecting Brucella infection, i.e., a serum and blood cell synchronous detection method. The detection method comprises two operation steps of serum sample detection and living blood cell sample detection and uses a supporting kit. The kit can be used for pretreatment of blood samples for clinically detecting Brucella in vitro. The serum and blood cell synchronous detection method can be used for early clinical rapid diagnosis of Brucella infection and medication guidance in the treatment process, and can also be used for prognosis, epidemiological survey of brucellosis, etc. The present invention can also be used for early clinical rapid diagnosis of other intracellular parasitic infection.

Claims

1. A pretreatment kit for detecting Brucella infection, comprising Solution I, Solution II, Solution III, Solution IV, and Solution V, wherein Solution I is an aqueous solution containing 368 mg/42 mL of sodium chloride; Solution II is an aqueous solution containing 1 g/125 mL of sodium chloride, 25 mg/125 mL of potassium chloride, 177.5 mg/125 mL of disodium hydrogen phosphate and 33.75 mg/125 mL of potassium dihydrogen phosphate; Solution III is an aqueous solution containing 42.399 mg/35 mL of trihydroxymethyl aminomethane, 306.81 mg/35 mL of sodium chloride and 102.284 mg/35 mL of ethylenediamine tetraacetic acid; Solution IV is 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol or an aqueous solution containing 35 mg/0.35 mL of sodium dodecyl sulfate; and Solution V is double distilled water.

2. A method for detecting or diagnosing Brucella infection of a subject, comprising: isolating and extracting DNA from a sample containing living cells using the pretreatment kit of claim 1; detecting whether a Brucella DNA is present in the extracted DNA; and diagnosing as having the Brucella infection when the Brucella DNA is present in the extracted DNA.

3. The method according to claim 2, further comprising enriching the living cells, wherein the sample is a peripheral blood.

4. The method according to claim 2, wherein the living cells are blood cells.

5. The method according to claim 2, further comprising detecting whether the Brucella DNA is in a body fluid or the living cells of the sample.

6. The method according to claim 2, wherein the method detects Mycobacterium tuberculosis infection or other intracellular parasitic infection.

7. The method according to claim 5, wherein the body fluid is serum, and the living cells are blood cells, and the serum and the living cells are from a peripheral blood taken from the subject.

8. The method according to claim 7, wherein the blood cells are living monocytes in the peripheral blood.

9. The method according to claim 2, wherein isolating and extracting the Brucella DNA comprises: taking 0.2 mL of serum into a 1.5 mL Ep tube; centrifuging at room temperature, 15000×g for 15 minutes to obtain a first pellet and a first supernatant; discarding the first supernatant; adding 0.2 mL of Solution V to the 1.5 mL Ep tube; shaking; centrifuging at room temperature, 15000×g for 10 minutes to obtain a second pellet and a second supernatant; discarding the second supernatant; adding 20 μL of Solution V to the 1.5 mL Ep tube; shaking, and instantaneously centrifuging; heating at 100° C. for 10 minutes; placing in ice or in a −20° C. refrigerator for 2 minutes; centrifuging at room temperature, 15000×g for 10 seconds to obtain a third supernatant; and sucking 15 or 10 μL of the third supernatant as a template for PCR.

10. The method according to claim 2, wherein isolating and extracting the Brucella DNA comprises: taking 1 mL of peripheral blood into a tube; adding 1 mL of Solution I into the tube and mixing uniformly to obtain a mixture; spreading the mixture in wells of a cell culture plate, and then incubating at 37° C. for 1 hour to obtain a sediment and a blood supernatant; discarding the blood supernatant in the wells, and washing the wells with Solution II until there is no red color; adding 1 mL of Solution II to the wells of the cell culture plate, repeatedly blowing, and transferring the sediment including living monocytes at the bottom of the wells to a 1.5 mL Ep tube; centrifuging the Ep tube at 4° C., 500×g for 5 minutes to obtain a first pellet and a first supernatant; discarding the first supernatant; adding 800 μL of Solution III to the first pellet, and mixing thoroughly; adding 8 μL of Solution IV, and mixing thoroughly; incubating at 55° C. for 1 hour; centrifuging the Ep tube at room temperature, 8000 rpm for 10 minutes to obtain a second pellet and a second supernatant; discarding the second supernatant; adding 800 μL of Solution V, and mixing thoroughly; centrifuging at room temperature, 8000 rpm for 10 minutes to obtain a third pellet and a third supernatant; discarding the third supernatant; adding 20 μL of Solution V, shaking, and mixing thoroughly; heating at 100° C. for 10 minutes; centrifuging at room temperature, 15000×g for 10 seconds to obtain a fourth supernatant; and sucking 15 or 10 μL of the fourth supernatant as a template for PCR.

11. The method according to claim 9, wherein the PCR is fluorescent quantitative PCR.

12. The method according to claim 10, wherein the PCR is fluorescent quantitative PCR.

Description

BRIEF DESCRIPTION OF THE DRAWING

(1) FIG. 1 shows a nucleic acid electrophoretogram of serum and blood cell PCR detection of Brucella infected patients, wherein 1, 2, 3, 4, 5 are five clinical patients, the left side is a serum Brucella DNA PCR detection map, and the right side is a corresponding blood cell Brucella DNA PCR detection map of the same patients; M is Marker; 1.5% agarose gel, 5 μL sample loading quantity; left and right arrows refer to target DNA bands.

DETAILED DESCRIPTION OF EMBODIMENTS

(2) The chemical reagents used in the following experimental operations were purchased from Sinopharm Chemical Reagent Co., Ltd., and the PCR kit and the agarose gel were purchased from Takara. All experiments were performed as usual in molecular biology experiments.

(3) Embodiment 1 Kit Components (Based on the Amount for a Kit to Detect 40 Brucella Infected Persons)

(4) Solution I (42 mL): 368 mg of sodium chloride; Solution II (125 mL): 1 g of sodium chloride, 25 mg of potassium chloride, 177.5 mg of disodium hydrogen phosphate, and 33.75 mg of potassium dihydrogen phosphate; Solution III (35 mL): 42.399 mg of trihydroxymethyl aminomethane, 306.81 mg of sodium chloride, and 102.284 mg of ethylenediamine tetraacetic acid, 25° C. pH 8.0; Solution IV (0.35 mL): TRITON™ X-100; Solution V (45 mL):double distilled water.

(5) Embodiment 2 Serum Sample Detection Steps

(6) Take 0.2 mL of serum into a 1.5 mL Ep tube; centrifuge at room temperature, 15000×g for 15 minutes; add 0.2 mL of Solution V to the 1.5 mL Ep tube; shake; centrifuge at room temperature, 15000×g for 10 minutes; discard the supernatant; add 20 μL of Solution V to the 1.5 mL Ep tube; shake, and instantaneously centrifuge; heat at 100° C. for 10 minutes; place in ice or in a −20° C. refrigerator for 2 minutes; centrifuge at room temperature, 15000×g for 10 minutes; and suck 15 (10) μL of supernatant as a template for PCR (including fluorescent quantitative PCR).

(7) Embodiment 3 Blood Cell Sample Detection Steps

(8) Take 1 mL of peripheral blood; add 1 mL of Solution I and mix uniformly; spread on a 6-well cell culture plate; incubate at 37° C. for 1 hour; wash with Solution II thoroughly (no red), and discard the supernatant; add 1 mL of Solution II to holes of the cell culture plate, repeatedly blow, and wash away the sediment (monocytes) at the bottom of the wells to a 1.5 mL Ep tube; centrifuge at 4° C., 500×g (about 1000 rpm) for 5 minutes; discard the supernatant; add 800 μL of Solution III, and mix thoroughly; add 8 μL of Solution IV, and mix thoroughly; incubate at 55° C. for 1 hour; centrifuge at room temperature, 8000 rpm for 10 minutes; discard the supernatant; add 800 μL of Solution V, and mix thoroughly; centrifuge at room temperature, 8000 rpm for 10 minutes; discard the supernatant; add 20 μL of Solution V, shake, and mix thoroughly; heat at 100° C. for 10 minutes; centrifuge at room temperature, 15000×g for 10 seconds; suck 15 (10) L of supernatant as a template for PCR (including fluorescent quantitative PCR).

(9) Embodiment 4 PCR Detection Results of a Small Amount of Samples Using the Above Reagents and Methods

(10) Serum and blood cell samples were collected from outpatients at Tongliao Endemic Disease Prevention and Treatment Stations from 8 Dec. 2016 to 5 Jan. 2017. The two kinds of samples were respectively subjected to the serum sample detection and blood cell sample detection steps of the present invention, and Brucella genomic DNA was extracted by using the kit of the present invention as a PCR template for PCR.

(11) Taking BCSP31 as a target gene, the primer sequences include B4 having the sequence of TGGCTCGGTTGCCAATATC (SEQ ID NO.: 1) and B5 having the sequence of CGCTTGCCTTTCAGGTCTG (SEQ ID NO.: 2).

(12) PCR system: B4 (20 μmol/L), 0.5 μL; B5 (20 μmol/L), 0.5 μL; dNTP Mixture (2.5 mmol/L each), 2 μL; 10×Buffer (Mg.sup.2+ plus), 2.5 μL; Taq DNA Polymerase (5 U/μL), 0.125 μL; template DNA, 15 μL or 10 μL of DNA extracted using the kit of the present invention; add the balance of double distilled water to 25 μL.

(13) PCR condition: DNA pre-denaturation at 90° C. for 5 minutes; denaturation at 90° C. for 1 minute, annealing at 53° C. for 1 minute, 72° C. extension for 1 minute, 40 cycles; 72° C. for 10 minutes; 4° C. cooling.

(14) FIG. 1 shows a nucleic acid electrophoretogram of serum and blood cell PCR detection of Brucella infected patients. It can be seen from the nucleic acid electrophoretogram of PCR detection that all 5 patients were infected with Brucella. By detection, patient 1 had negative serum and strongly positive blood cells; patient 2 had strongly positive serum and blood cells; patient 3 had strongly positive serum and negative blood cells; patient 4 had positive serum and blood cells, but the serum was extremely weak positive, it can be determined that the patient's serum contained trace Brucella, that is, the bacterial load of the serum was very low, the blood cells were strongly positive, it can be determined that the bacterial load of the patient's blood cells was high, and the patient can be diagnosed as a chronic brucellosis patient in a treatment phase in combination with the patient's other clinical manifestations; the conclusion of patient 5 was the same as that of patient 4, but from the position of the DNA band indicated by the right arrow, it can be seen that the Brucella load of the blood cells of patient 5 may be higher, the infected bacteria type may also be different from that of other patients, and further genetic identification is needed.

(15) Embodiment 5 Analysis on the Results of Serum and Blood Cell Synchronous Detection of 300 Brucellosis Patients Using the Above Reagents and Method, and the Results of Rose-Bengal Plate Agglutination and Tube Agglutination Test

(16) 1. Samples were collected from 8 Dec. 2016 to 26 May 2017, in a total of 300 people including clinic suspected cases in hospitals of Tongliao City and people who had a history of exposure to Brucella and volunteered to receive brucellosis antibody detection and physical examination in Tongliao Endemic Disease Prevention and Treatment Stations.

(17) 2. Detection reagents, including brucellosis rose-bengal plate agglutination antigen reagent, brucellosis tube agglutination antigen reagent, and positive serum and negative serum of brucellosis, were purchased from the National Institute for Communicable Disease Control and Prevention, and were all within the validity period.

(18) 3. Samples were collected using blood collection tubes with a separation gel coagulant, 5 mL of whole blood of test objects was taken on seat, the whole blood was centrifuged after 30 minutes to separate serum, and the serum was stored at 4° C. for later use.

(19) 4. Materials and methods required for serum and blood cell synchronous detection are the same as those in Embodiment 4.

(20) 5. SAT test was operated according to the daily standard operating procedures, the serum to be tested was diluted with 0.5% carbolic acid normal saline, 0.5 mL of liquid was reserved in each tube, then 0.5 mL of 10 times diluted brucellosis tube agglutination antigen reagent was added, and the final serum dilution was 1:25, 1:50, 1:100, 1:200. The same method was used to make negative and positive control tubes and turbidimetric tubes. After fully shaking, the serum was incubated in a 37° C. incubator for 20 to 22 hours, then taken out, and placed at room temperature for 2 hours to observe the results.

(21) 6. RBPT test was operated according to the daily standard operating procedures, 30 μL of serum to be tested was sucked onto a clean glass slide with a pipette, the tip was discarded and replaced with a new one to suck 30 μL of rose-bengal plate agglutination antigen reagent to mix thoroughly with the serum, and results were observed within 5 minutes.

(22) 7. The results were judged according to the 2007 edition of WS269-2007 Brucellosis Diagnosis Criteria issued by the Ministry of Health of the People's Republic of China. SAT recorded serum dilution and transparency in the form of (−), 1:25 (+), 1:25 (++) . . . 1:200 (++++); RBPT recorded the degree of agglutination in the form of (−), (+−), (+), (++), (+++) and (++++). The results of contrast analysis were shown in Table 1, and the test results of 300 cases were shown in Table 2.

(23) TABLE-US-00001 TABLE 1 Contrast analysis results of 300 cases Blood cell PCR (+) 225 cases Antibody (+) 167 cases (55.7%) Confirm 279 cases of (75%) Antibody (−) 58 cases (19.3%) brucellosis (93%) Serum PCR (+) 153 cases (51%) Antibody (+) 130 cases (43.3%) Antibody (−) 23 cases (7.7%) Blood cell and serum simultaneous Antibody (+) 93 cases (31%) PCR (+) 99 cases (33%) Antibody (−) 6 cases (2%) Blood cell PCR (+) or serum Antibody (+) 204 cases (68%) PCR (+) 279 cases (93%) Antibody (−) 75 cases (25%) Blood cell and serum simultaneous Antibody (+) 20 cases (6.7%) Exclude 21 cases of PCR (−) 21 cases (7%) Antibody (−) 1 case (0.3%) brucellosis (7%)

(24) Conclusion:

(25) In serum and blood cell synchronous detection, brucellosis confirmation rate 279/300=93%, brucellosis exclusion rate 21/300=7%.

(26) In antibody test, brucellosis confirmation rate 174/300=58%.

(27) TABLE-US-00002 TABLE 2 Test results of 300 cases PCR Clinic Result Date No. Gender Age Address Blood Cells Serum SAT RBPT December 8 1 male 34 Kequ + + − − December 9 2 male 35 Zuozhong + + 1:25++ + 3 male 43 Houqi + +   .sup.  1:200++++ + 4 male 24 Kequ − + − − 5 male 48 Houqi + +   .sup.  1:100++++ + 6 female 37 Kequ + −   .sup.  1:200++++ + December 19 7 male 71 Kequ + −  1:200++ + 8 male 34 Houqi + −  1:200++ + 9 male 39 Houqi + − − − 10 female 29 Zuozhong + − − − 11 male 40 Heilongjiang + − 1:25++ + 12 male 20 Heilongjiang + − 1:50++ + 13 female 40 Heilongjiang + + − − 14 male 46 Kequ − − 1:25++ + 15 female 32 Development + − − − District 16 female 53 Zuozhong + −  1:200++ + 17 female 46 Zuozhong + − 1:50++ + 18 male 26 Liaoning + − − − 19 female 44 Heilongjiang − − 1:50++ + 20 male 42 Heilongjiang + − 1:50++ + 21 male 59 Kailu + −  1:200++ + December 21 22 male 60 Houqi + − 1:50++ + 23 female 23 Houqi + −   .sup.  1:200++++ + 24 male 34 Houqi + − − + 25 female 44 Zuozhong + − 1:50++ + December 26 26 male 57 Kequ + − − − 27 female 57 Youzhong + −  1:100++ + 28 female 44 Houqi + − − − 29 female 44 Liaoning + − − − 30 female 54 Kequ + − − − 31 female 34 Houqi + − − − December 27 32 male 51 Xing'an + − − − League 33 female 28 Kequ + − − − 34 female 33 Zhaqi + −  1:200++ + 35 female 54 Liaoning + −  1:100++ + 36 male 21 Liaoning + −  1:200+++ + 37 male 39 Xing'an − − − − League 38 male 36 Zuozhong + −  1:200+++ + 39 male 54 Kequ − − 1:25++ + 40 male 48 Zuozhong + −  1:100++ + 41 male 44 Heilongjiang + +  1:200+++ + 42 male 54 Jilin + + − − 43 female 31 Zuozhong + +  1:200++ + 44 female 47 Zuozhong + + − − December 28 45 female 66 Zuozhong + − 1:50++ + 46 female 38 Zuozhong + −  .sup. 1:25++++ + 47 male 26 Chifeng + −  1:50+++ + 48 female 51 Zhaqi + − 1:100+ + 49 male 39 Xing'an + − 1:50+  + League 50 male 35 Kequ + − − + 51 male 36 Kequ + − − − 52 male 43 Houqi + − 1:50+  + January 5 53 male 43 Houqi + −  1:100++ + 54 male 45 Houqi + −  1:100++ + 55 male 23 Houqi + − 1:25++ + 56 male 65 Kequ + −  .sup. 1:25++++ + 57 male 25 Kailu + − − − 58 male 42 Kailu + − 1:25++ + 59 male 31 Zhaqi + −   .sup.  1:100++++ + 60 male 31 Zhaqi + − − − 61 male 57 Zuozhong + −  1:100++ + 62 female 54 Development + − 1:25+  + District 63 male 42 Naiman + + − − 64 male 59 Jilin + − − − 65 male 38 Kailu + + − − 66 female 39 Zuozhong + − 1:25+  + 67 male 39 Houqi + − − − 68 female 41 Xing'an + − − − League 69 female 66 Kailu + − − − May 22 70 male 30 Development − − 1:25++ + District 71 female 27 Houqi − + − − 72 female 32 Development − − 1:25++ + District 73 male 38 Jilin − − 1:25++ + 74 male 21 Zuozhong − +   .sup.  1:200++++ + 75 male 48 Zuozhong + − 1:50++ + 76 female 26 Kequ − + − − 77 female 53 Houqi + − 1:25++ + 78 male 39 Houqi − + 1:50++ + 79 male 42 Kequ + +  1:100+++ + 80 male 26 Zuozhong + −  1:100++ + 81 male 44 Zuozhong − − 1:25++ + 82 female 44 Kequ + +  1:200+++ + 83 male 29 Zuozhong − + − − 84 female 30 Kulun − − 1:50+  + 85 male 44 Kequ − + 1:50++ + 86 male 42 Kequ + +   .sup.  1:200++++ + 87 male 30 Zuozhong + −  1:100++ + 88 male 47 Liaoning + −  1:100++ + 89 male 35 Development + −  1:100+++ + District 90 male 42 Xing'an + +  1:50+++ + League 91 male 33 Kequ + +   .sup.  1:200++++ + 92 male 59 Zhaqi + −   .sup.  1:200++++ + 93 female 33 Development − + 1:50++ + District 94 male 42 Liaoning − +  1:25+++ + 95 male 36 Zuozhong + +  1:200+++ + 96 female 57 Kequ + − − − 97 male 49 Houqi + +  1:200+++ + 98 male 51 Zuozhong + +  1:50+++ + 99 male 58 Zuozhong + −  1:200++ + 100 male 43 Kequ + −  1:100+++ + 101 female 41 Zuozhong − − 1:25++ + 102 male 48 Zhaqi + +  1:200++ + 103 male 52 Liaoning + +  1:200+++ + 104 female 46 Zuozhong + +   .sup.  1:200++++ + 105 female 57 Houqi + − − − 106 female 34 Kequ − − 1:25++ + 107 male 56 Kequ − +  1:50+++ + 108 male 47 Jilin + +   .sup.  1:200++++ + 109 male 51 Zuozhong + +  1:200+++ + 110 male 51 Houqi + − − − 111 male 51 Hebei + +  1:100++ + 112 female 44 Zuozhong − + 1:50++ + 113 female 53 Kequ + − − − 114 male 45 Zuozhong + −  1:100++ + 115 female 64 Zuozhong + −  1:200++ + 116 male 60 Kequ − − 1:50+  + May 23 117 male 47 Zuozhong + + 1:50++ + 118 female 48 Kequ + +   .sup.  1:200++++ + 119 male 56 Kequ + +   .sup.  1:200++++ + 120 male 28 Houqi − + − − 121 female 54 Kequ + + 1:100+ + 122 male 43 Xing'an + −  1:200+++ + League 123 male 53 Xing'an + −  1:200++ + League 124 female 52 Houqi − + − − 125 male 68 Zuozhong − + − − 126 male 41 Kequ − + 1:50++ + 127 male 37 Kequ − + 1:100+ + 128 male 24 Kequ + +   .sup.  1:200++++ + 129 male 27 Heilongjiang + + 1:50+  + 130 male 24 Kailu + + 1:100+ + 131 male 31 Houqi + +   .sup.  1:200++++ + 132 female 50 Heilongjiang + − − − 133 male 37 Heilongjiang − + − − 134 male 49 Houqi − +  1:100++ + 135 male 29 Houqi + +  1:200++ + 136 female 28 Kequ − + − − 137 male 45 Houqi − +  1:50+++ + 138 male 51 Development − − 1:50+  + District 139 male 26 Kequ − +  1:100++ + 140 male 33 Zuozhong + +  1:200++ + 141 male 44 Zuozhong − − − + 142 female 40 Zuozhong + − − − 143 female 44 Zuozhong + −  1:200++ + 144 male 47 Development + −  1:100+++ + District 145 male 37 Liaoning − − 1:50+  + 146 female 26 Heilongjiang − + − − 147 male 58 Development + +   .sup.  1:200++++ + District 148 male 29 Liaoning + +  1:200++ + 149 male 30 Kequ + +  1:200++ + 150 male 5 Heilongjiang − −  .sup. 1:25++++ + 151 male 41 Kequ + −  1:200++ + 152 male 29 Zuozhong − +  1:100++ + 153 female 38 Kailu − − 1:25++ + 154 male 54 Kequ + −  1:100+++ + 155 male 43 Zuozhong + −  1:100+++ + 156 male 42 Zuozhong + +   .sup.  1:200++++ + 157 female 33 Houqi + − − − 158 female 49 Houqi + − − − 159 male 33 Houqi + −  1:100++ + 160 male 58 Zuozhong − + 1:50++ + 161 male 42 Houqi − − 1:25++ + 162 male 55 Development + +  1:50+++ + District 163 male 31 Houqi − + 1:50++ + 164 male 44 Houqi + +   .sup.  1:200++++ + 165 male 54 Houqi + +  1:200++ + 166 female 30 Zuozhong − + − − 167 male 32 Kequ + − − − 168 male 33 Liaoning + +   .sup.  1:200++++ + 169 female 60 Youzhong + − − − 170 male 57 Liaoning + +  1:200+++ + 171 male 57 Kulun + +  1:100+++ + 172 male 41 Zhaqi + −  1:200+++ + 173 male 27 Liaoning + + 1:50++ + 174 male 21 Chifeng + + 1:50++ + 175 male 28 Houqi − − 1:25++ + 176 male 47 Liaoning + − − − 177 male 40 Kequ + − − − 178 male 31 Development + +  1:100++ + District 179 female 45 Houqi + + 1:50++ + 180 male 24 Houqi − +  1:200+++ + 181 male 53 Houqi − + 1:25++ + 182 female 43 Xing'an + − − − League 183 male 34 Liaoning + + 1:50++ + 184 male 34 Development + + 1:50++ + District May 24 185 male 43 Kequ + − − − 186 male 20 Zuozhong − +  1:200++ + 187 female 54 Zhaqi + − − − 188 male 39 Zuozhong + −   .sup.  1:200++++ + 189 female 42 Xing'an + +   .sup.  1:200++++ + League 190 male 50 Kequ − +  1:100++ + 191 female 56 Liaoning + − − − 192 female 36 Liaoning + 1:25++ + 193 male 60 Zuozhong + + 1:50++ + 194 female 59 Kequ + +  1:200+++ + 195 male 64 Xing'an + +   .sup.  1:200++++ + League 196 male 37 Zuozhong − +  1:100+++ + 197 female 27 Zuozhong − + 1:25++ + 198 male 24 Zhaqi + −   .sup.  1:200++++ + 199 female 50 Kailu + −  1:200+++ + 200 male 22 Kequ − + − − 201 male 29 Zuozhong + − 1:25++ + 202 male 11 Kailu + +   .sup.  1:200++++ + 203 male 59 Zuozhong + +  1:100++ + 204 female 40 Heilongjiang + − − − 205 male 62 Houqi + +   .sup.  1:200++++ + 206 female 46 Kequ + +  1:200+++ + 207 male 37 Jilin + − 1:25++ + 208 female 31 Zuozhong − + − − 209 male 40 Houqi + +   .sup.  1:200++++ + 210 male 61 Zhaqi + +   .sup.  1:200++++ + 211 female 57 Zuozhong + − − − 212 male 33 Zuozhong + − 1:25++ + 213 female 45 Shandong + +  1:200+++ + 214 male 40 Houqi + +  1:200++ + 215 female 44 Kulun − + 1:50++ + 216 male 26 Zuozhong − + 1:25++ + 217 male 50 Chifeng + − − − 218 male 29 Kailu + +  1:200+++ + 219 male 24 Liaoning − +  1:200++ + 220 male 37 Zhaqi + − 1:25++ + 221 female 40 Xing'an + − − − League 222 male 44 Kequ + +  1:100++ + 223 female 71 Kequ + − − − 224 male 55 Houqi + − 1:25++ + 225 male 31 Kailu + +   .sup.  1:200++++ + 226 female 61 Liaoning + + 1:50++ + 227 male 61 Zhaqi + +  1:100++ + 228 female 45 Houqi + − − − 229 male 46 Houqi + − − − 230 male 32 Kailu − +  1:100++ + 231 female 24 Zuozhong + +  1:100++ + 232 male 30 Zuozhong + +  1:200++ + May 25 233 female 40 Houqi − + 1:25++ + 234 female 32 Kequ + − − − 235 male 31 Kequ + + 1:50++ + 236 female 49 Kailu + − − − 237 male 30 Zuozhong + +  1:200+++ + 238 female 29 Naiman + + 1:50++ + 239 female 50 Kailu + − − − 240 male 37 Zuozhong − + 1:25++ + 241 male 45 Kequ + +   .sup.  1:200++++ + 242 female 28 Kequ + +  1:200+++ + 243 male 57 Development + − − − District 244 female 26 Naiman − +  1:25+++ + 245 female 36 Zuozhong + +  1:200+++ + 246 male 57 Zuozhong + − 1:50++ + 247 male 27 Chifeng − +  1:200+++ + 248 female 43 Jilin − + − − 249 male 31 Xilinhot + +  1:200+++ + 250 male 45 Liaoning + +  1:100++ + 251 female 39 Kailu + − − − 252 male 54 Heilongjiang + −   .sup.  1:200++++ + 253 male 56 Kequ + +   .sup.  1:200++++ + 254 male 39 Zuozhong + − − − 255 male 38 Houqi + − 1:25++ − 256 female 50 Houqi + + 1:50++ + 257 male 33 Zuozhong − + 1:25+  + 258 male 54 Xing'an + − − − League 259 female 55 Kequ + +   .sup.  1:200++++ + 260 male 32 Zuozhong + +  1:200++ + 261 male 43 Kequ + − 1:25+  + 262 male 68 Zuozhong + − 1:200+ + 263 female 34 Xing'an − + − − League 264 male 26 Kailu − +   .sup.  1:200++++ + 265 male 44 Zuozhong + +   .sup.  1:200++++ + 266 male 51 Zuozhong + − 1:50++ + 267 female 49 Kequ + − 1:25+  + 268 female 51 Zuozhong + − − − 269 male 30 Zuozhong + +  1:100++ + 270 male 48 Houqi + − 1:50++ + 271 male 75 Kailu − +  1:200++ + May 26 272 male 50 Naiman + − − − 273 male 40 Naiman − + 1:50+  + 274 male 45 Houqi + −  1:200++ + 275 female 61 Zhaqi + − − − 276 male 37 Zhaqi − + − − 277 male 42 Houqi − +  1:100++ + 278 female 55 Kequ + +   .sup.  1:100++++ + 279 male 45 Development + + 1:100+ + District 280 female 40 Zuozhong − − − + 281 female 54 Zuozhong − − 1:25+  + 282 male 40 Houqi − +  1:200++ + 283 male 60 Zhaqi + +   .sup.  1:200++++ + 284 male 26 Zhaqi + +  1:200+++ + 285 male 25 Houqi + +   .sup.  1:200++++ + 286 female 51 Naiman + − 1:25+  + 287 male 35 Naiman − + − − 288 male 65 Kequ + +  1:100++ + 289 male 56 Kequ + +  1:200+++ + 290 male 59 Kailu + − − − 291 male 36 Kequ + − 1:50+  + 292 female 38 Zuozhong + + 1:50++ + 293 male 29 Houqi + +   .sup.  1:200++++ + 294 male 56 Development + +  1:100+++ + District 295 male 51 Liaoning + + 1:100+ + 296 female 59 Youqi + +  1:200+++ + 297 male 37 Youqi + +   .sup.  1:200++++ + 298 female 23 Xing'an − +   .sup.  1:200++++ + League 299 male 63 Kequ + + 1:100+ + 300 male 47 Zhaqi + + 1:50++ +