WS/T 633-2018Detection of Babesia spp. - Identification of species by nucleic acid method (English PDF)

4.1 High speed centrifuge

The maximum relative centrifugal force is 12 000 × g.

4.2 Vortex

The stepless speed regulation range is from.200 r/min to 3 000 r/min.

4.3 PCR instrument

The temperature range is from 4 ° C to 99 ° C, the temperature is precisely 0.1 ° C, and the hot lid is 105 ° C.

4.4 Microwave oven

4.5 Electrophoresis

The voltage is 5 V to 5 000 V and the current is 1 mA to 500 mA.

4.6 Gel Imaging System

The lens resolution (H × V) is 1 360 × 1 024 and the light source is transmissive UV.

5.1 Molecular biology reagents

Commercially available products are selected for Taq enzymes, deoxyribonucleoside triphosphates (dNTPs), molecular mass indicators, and nucleic acid extraction kits.

5.2 Chemical reagents

Tris-acetate running buffer (TAE), agarose gel, 6 x loading buffer (see Appendix B).

5.3 Primers

Specific primers for amplifying the 18S ribosomal RNA (18S rRNA) gene and the transcribed spacer (ITS) gene of Babesia (see Appendix) C).

5.4 Reference materials

The positive control was genomic DNA of the peabody mjr strain of the hamster.

The blank control was deionized water.

The negative control is whole blood genomic DNA of healthy individuals.

6.1 Sample

The sample is an anticoagulant sample for clinical diagnosis or suspected Babesia case.

6.2 Identification of insect nucleic acid

6.2.1 Nucleic acid extraction

Genomic DNA extraction uses a commercial whole blood DNA extraction kit (see Appendix B).

6.2.2 18S rRNA gene amplification See Appendix C.

6.2.3 ITS gene amplification

See Appendix C.

6.2.4 Result determination

6.2.4.1 Electrophoresis analysis

6.2.4.1.1 Electrophoresis

Take 5 µL of the second round of PCR product and mix with 1 µL of 6× loading buffer and apply to 1.0% agarose gel containing ethidium bromide substitute.

In electrophoresis at 5 V/cm in 1×TAE buffer for about 40 min, stop electrophoresis when bromophenol blue reaches the bottom, using a gel imaging system or UV analyzer analysis.

6.2.4.1.2 Judgment of PCR results

6.2.4.1.2.1 PCR amplification of the 18S rRNA gene of Babesia, the amplification fragment with a specificity of about 400 bp, blank pair no bands appeared in the negative and negative controls, and the PCR results were positive. PCR amplification of Babesia 18S rRNA gene, no size of 400 bp left the right-specific amplified fragment, the blank control and the negative control did not show a band, and the PCR result was negative.

6.2.4.1.2.2 PCR amplification of the IBS gene of Babesia, the emergence of specific amplified fragments ranging from 700 bp to 2 100 bp, blank pair no bands appeared in the negative and negative controls, and the PCR results were positive. PCR amplification of Babesia ITS gene, no specific amplified fragment, empty There were no bands in the white control and the negative control, and the PCR results were negative.

6.2.4.2 Sequencing analysis

Positive PCR amplification products were sequenced in both directions and the sequences were subjected to BLAST alignment analysis (see Appendix C).

6.2.4.3 Identification of species

According to the electrophoresis results of the PCR amplification products and the sequencing results, the species are comprehensively determined (see Appendix C).

Appendix A

(informative appendix) Pathogenic biology A.1 Classification Babesia spp. belongs to Apicomplexa, Sporozoa, and P.

(Piroplasmasina), Piroplasmida, Babesia (Babesia) Babesia. Currently More than 100 species of Babesia have been identified that can infect a variety of mammals and birds such as cattle, horses, sheep, and dogs. In more than 100 species of Babe, both the main types of Babesia known to infect humans are. B. microti, B. divergens, Dunkenba.

B. duncani (also known as WA1) and B. venatorum (also known as EU1). Different Babeite species See Table A.1 for a comparison of clinical symptoms.

A.2 distribution

The disease is endemic.

In the United States, cases of Babesia are mainly found in Massachusetts, New York, Connecticut, California, and Rhode Island.

In Europe, cases of Babesia in France, Portugal, Italy, Poland, Norway and other places have occurred. Also, in Egypt, Mexico, Cases of Babesia have also been reported in Australia, Japan, South Korea, Pakistan and South Africa.

In China, Yunnan, Henan, Xinjiang, Shandong, Shaanxi, Zhejiang, Jiangsu, Guangxi, Fujian, Taiwan, Hong Kong and other places have people Case reports of beetle disease.

Table A.1 Comparison of clinical symptoms caused by different Babesia species

Insect species geographical distribution media name latency clinical characteristics other Field mouse (B.microti) Europe, the United States, Japan, China, Egypt, etc.

Shoulder blade hard, Oval hard, Full ditch Dan Min hard 1 week to 6 weeks, even 3 More than a month Most patients have mild symptoms and only appear once Sexual fever symptoms; severe cases of malaria Symptoms, including fever, chills, muscle pain, poor Blood, weakness, hepatosplenomegaly, hemolytic anemia, etc.

Generally over 50 years old Elderly susceptibility Baba (B.divergens) European scorpion hard sputum 1 week ~ 3 weeks suddenly onset, hemoglobinuria, jaundice, shock-like which performed Spleen resection and immunity Defective Duncan Babe (B.duncani) Europe, the United States Dan Min is hard, Shoulder blade 3 weeks to 2 months of fever, chills, muscle pain, anemia, weakness, Hepatosplenomegaly, hemolytic anemia, lasting weeks or more Spleen resection is easy Sense; easy transfusion Babe (B.venatorum) Europe, China Dice hard, Oval hard it is not clear that most patients have mild symptoms and only appear once Sexual fever symptoms; severe cases of anemia, blood red Proteinuria, jaundice and other symptoms Immune dysfunction or splenectomy patients Susceptible BB Appendix B (informative appendix) Reagent preparation B.1 Preparation of reagents B.1.1 Preparation of Tris- acetic acid buffer solution (TAE) Preparation of 50× TAE. tris(hydroxymethyl)aminomethane (Tris base) 242 g, glacial acetic acid 57.1 mL, 0.5 mol/L EDTA (pH 8.0) 100 mL, add deionized water to a volume of 1 000 mL, mix and mix at 4 ° C for later use.

B.1.2 Preparation of 1× TAE liquid 50 × TAE 20 mL with distilled water to a volume of 1 000 mL, mix and set aside.

B.1.3 Preparation of 1.0% agarose gel Agarose 1.0 g plus 1× TAE to a volume of 100 mL. After the microwave oven is completely melted, the solution is cooled to 60 °C, plus 10 5 µL of mg/mL ethidium bromide substitute (final concentration 0.5 µg/mL), gently mix and prepare a gel.

B.1.4 Preparation of 6× loading buffer Bromophenol blue 0.25 g, sucrose 40 g, add distilled water to 100 mL. Store at 4 °C for later use.

B.2 DNA extraction

Take 100 µL of blood sample and extract the genomic DNA using the commercially available whole blood sample DNA extraction kit. The procedure is as described in the kit.

The book is carried out.

Appendix C

(normative appendix) PCR experiments, sequencing results analysis and standard reference sequences C.1 PCR amplification primer sequence the primers for identification of Babesia species are shown in Table C.1.

Table C.1 Babesia species identification primers

Primer Primer Name Primer Sequence a Fragment Size b References 18S rRNA Lateral primer Bab 5 5'-AATTACCCAATCCTGACACAGG-3' Around 400 bp [3] Bab 8 5'-TTTCGCAGTAGTTCGTCTTTAACA-3' 18S rRNA Inner primer Bab 6 5'-GACACAGGGAGGTAGTGACAAGA-3' Bab 7 5'-CCCAACTGCTCCTATTAACCATTAC-3' ITS Lateral primer ITS-F3 5'-GGTGGTGCATGGCCG-3' 700 bp to 2 100 bp [4] ITS-R3 5'-T(A/T)GCGCTTCAATCCC-3' ITS Inner primer ITS-F4 5'-GAGAAGTCGTAACAAGGTTTCCG-3' ITS-R4 5'-GCTTCACTCGCCGTTACTAGG-3' the a18S rRNA gene primer sequence and PCR reaction were taken from WS/T 564.

b Different insect species have different sizes.

C.2 Primer preparation the primers were formulated into deionized water to prepare a stock solution of 100 µmol/L. Then take a part and add the corresponding volume of deionized water thin Released into 20 µmol/L working solution for use.

C.3 18S rRNA PCR reaction system and reaction conditions C.3.1 18S rRNA PCR first round reaction C.3.1.1 The first round of amplification was performed with primers Bab 5 and Bab 8, and the reaction system was 50 µL.

Taq enzyme (5 U/µL) 0.25 µL 10×PCR buffer (containing 15 mmol/L Mg2) 5 µL 25 mmol/L dNTPs 4 µL 20 µmol/L Bab 5 1.5 µL 20µmol/L Bab 8 1.5 µL Genomic DNA template (about 70 ng) 2 µL Deionized water 35.75 µL 50 µL in total Positive, negative and blank controls were required for the PCR reaction.

C.3.1.2 PCR conditions 95 °C 5 min; 95 °C 45 s, 55 °C 45 s, 72 °C 1 min, a total of 35 cycles; 72 °C 7 min; 4 °C infinity.

C.3.2 18S rRNA PCR second round reaction C.3.2.1 Take 2 µL of the first round of PCR product as template and carry out the second round of amplification with primers Bab 6 and Bab 7. The reaction system is 50 µL.

Taq enzyme (5 U/µL) 0.25 µL 10×PCR buffer (containing 15 mmol/L Mg2) 5 µL 25 mmol/L dNTPs 4 µL 20 µmol/L Bab6 1.5 µL 20 µmol/L Bab7 1.5 µL First round of PCR product 2 µL Deionized water 35.75 µL 50 µL in total C.3.2.2 PCR conditions 95 °C 5 min; 94 °C 45 s, 55 °C 45 s, 72 °C 1 min, a total of 35 cycles; 72 °C 7 min; 4 °C infinity.

C.4 ITS PCR reaction system and reaction conditions C.4.1 ITS PCR first round reaction C.4.1.1 The first round of amplification was performed with primers ITS-F3 and ITS-R3 with a reaction system of 50 µL.

Taq enzyme (5 U/µL) 0.25 µL 10×PCR buffer (containing 15 mmol/L Mg2 ) 5 µL 25 mmol/L dNTPs 4 µL 20 µmol/L ITS-F3 1.5 µL 20 µmol/L ITS-R3 1.5 µL Genomic DNA template (about 70 ng) 2 µL Deionized water 35.75 µL 50 µL in total Positive, negative and blank controls were required for the PCR reaction.

C.4.1.2 PCR conditions 95 °C 5 min; 94 °C 30 s, 50 °C 30 s, 72 °C 2 min, a total of 35 cycles; 72 °C 10 min; 4 °C infinity.

C.4.2 ITS PCR second round reaction C.4.2.1 Take 2 µL of the first round of PCR product as template and carry out the second round of amplification with primers ITS-F4 and ITS-R4. The reaction system is 50 µL.

Taq enzyme (5 U/µL) 0.25 µL 10×PCR buffer (containing 15 mmol/L Mg2) 5 µL 25 mmol/L dNTPs 4 µL 20 µmol/L ITS-F4 1.5 µL 20 µmol/L ITS-R4 1.5 µL First round of PCR product 2 µL Deionized water 35.75 µL 50 µL in total C.4.2.2 PCR conditions 95 °C 5 min; 94 °C 30 s, 60 °C 30 s, 72 °C 2 min, a total of 35 cycles; 72 °C 10 min; 4 °C infinity.

Note. PCR experiments are in accordance with WS/T 230.

C.5 Analysis of sequencing results Sequence homology analysis using Blastn (https.//blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn =BlastSearch

a) The 18S rRNA gene sequence is more than 96% similar to the Babesia 18S rRNA sequence in GenBank, or the ITS gene sequence is the similarity of ITS sequence in GenBank is more than 95%, which is determined as Babesia;

b) The 18S rRNA gene sequence is more than 99% similar to the 18S rRNA sequence of the Babesia variabilis, or the ITS gene sequence and the voles the similarity of the IBS sequence of Babesia was over 98%, which was determined to be Babesia variabilis;

c) The 18S rRNA gene sequence is more than 99% similar to the Babesia 18S rRNA sequence, or the ITS gene sequence is different from the difference the similarity of the IBS sequence of the beetle was over 98%, which was judged to be a divergent Babesia;

d) The 18S rRNA gene sequence is more than 99% similar to the Duncan Babesia 18S rRNA sequence and is judged to be Duncan Babesia;

e) The 18S rRNA gene sequence was more than 99% similar to the 18S rRNA sequence of the Babe worm, and was identified as the hunter Babe.

C.6 standard reference sequence C.6.1 18S rRNA standard reference sequence C.6.1.1 Field mouse Babesia 18S rRNA gene reference sequence (reference sequence GenBank serial number KU204794, which is in the gene the starting position is 405-811) GACACAGGGAGGTAGTGACAAGAAATAACAATACAGGGCTTAAAGTCTTGTAATTGGAATGATGGGAATCTAAAC CCTTCCCAGAGTATCAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATT AAAGTTGTTGCAGTTAAGAAGCTCGTAGTTGAATTTCTGCCTTGTCATTAATCTCGCTTCCGAGCGTTTTTTTATTG ACTTGGCATCTTCTGGATTTGGTGCCTTCGGGTACTATTTTCCAGGATTTACTTTGAGAAAACTAGAGTGTTTCAAA CAGGCATTCGCCTTGAATACTACAGCATGGAATAATGAAGTAGGACTTTGGTTCTATTTTGTTGGTTATTGAGCCA GAGTAATGGTTAATAGGAGCAGTTGGG (407 bp) C.6.1.2 The reference sequence of the Babesia 18S rRNA gene (reference sequence GenBank serial number is KP745627, which is in this gene Starting position is 347-725) GACACAGGGAGGTAGTGACAAGAAATAACAATACAGGGCAATTGTCTTGTAATTGGAATGATGGTGACCTAAACC CTCACCAGAGTAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTA AACTTGTTGCAGTTAAAAAGCTCGTAGTTGAATTTTTGCGTGGTGTTAATATTGACTAATGTCGAGATTGCACTTCG CTTTTGGGATTTATCCCTTTTTACTTTGAGAAAATTAGAGTGTTTCAAGCAGACTTTTGTCTTGAATACTTCAGCAT GGAATAATAGAGTAGGACTTTGGTTCTATTTTGTTGGTTTGTGAACCTTAGTAATGGTTAATAGGAACGGTTGGG (379 bp) C.6.1.3 Duncan Babesia 18S rRNA gene reference sequence (reference sequence GenBank serial number is HQ289870, which is in the gene the starting position is 445-842) GACACCGTGAGGTAGTGACAAGAAATAACAATACAGGGCTTAAAGCTTTGTAATTGGAATGATGGGAATCCAAAC CCCTTCCAGAGTATCAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTA AACTTGTTGCAGTTAAAAAGCTCGTAGTTGAACTTCTGCCGCTTGGCCTTCGTTCCCCTTGGGGTTTCGTTCGCCTG GTGGCTTACCTCTGGCGGTGGTTCTCCATTTGCCAGTTTTACTTTGAGAAAATTAGAGTGTTTCAAGCAGGCTTTTG CCTTGAATACTTCAGCATGGAATAATAAAGTAGGACTTTGGTTCTATTTTGTTGGTTTCAGGACCAAAGTAATGGTT AATAGGAACAGTTGGG (398 bp) C.6.1.4 Orion Babesia 18S rRNA gene reference sequence (reference sequence GenBank serial number is KF724377, which is in this gene Starting position is 423-801) GACACAGGGAGGTAGTGACAAGAAATAACAATACAGGGCAATTGTCTTGTAAATGGAATGATGGTGACCTAAACC CTCACCAGAGTAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTA AACTTGTTGCAGTTAAAAAGCTCGTAGTTGAATTTCTGCGTTATCGAGTTATTGACTCTTGTCTTTAATCGATTTCG CTTTTGGGATTTATCCCTTTTTACTTTGAGAAAATTAGAGTGTTTCAAGCAGACTTTTGTCTTGAATACTTCAGCAT GGAATAATAGAGTAGGACTTTGGTTCTATTTTGTTGGTTTTTGAACCTTAGTAATGGTTAATAGGAACGGTTGGG (379 bp) C.6.2 ITS standard reference sequence C.6.2.1 The ITS standard reference sequence of the hamster Babesia (reference sequence GenBank serial number is AF510198, which is the starting position in this gene Set to 1-828) TTAAAGGAAGGAGAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTCTTATTAGAATTCTATGTA TCCGTTTGGGTACGCTGGCCCGTGCGCTTGGTCGCACCTTTTATAGTAGTGGCCTTGGACGTAGCGGCTGCAACTTG TTGGAGCCGCATCCCCGTGGTGGCACAACGCGTCGGAAGGCGGGTTTGTCCGCTATGGGGCGTTCTCACTCCCAAG GTATTTTCCCCGGGCCTCTGTGTTAGATCATTCTAACGGGCTTGGGGGCTATACTGTCATTCGCTGAGAAGTGCCCCC GTGGCTTTCTTGGCTGCTAACGTGGCTTTTCTCGGAAGGTCATGAGGAGCGATTTGTCTAACAACTACTACGAATATT AAACGAAACAAATTTTCAGCGGTGGATGTCTCGGCTCACACAACGATGAAGGACGCAGCGAAGTGCGATAATCATT GTGAATTGCAGAATTTAGCAAATCAACAGGTTTCTGAATGTATTGTACACACTGCCTCTGTTCGCAAGCAGTGCACC CATTTCAGCGCCTCTTAAAATCCTAAAGTATACCTTTTTCCAAGTATGCTTTGTGAGTGGTGCGCAACAGATTTTCTGT GTGTGCATTTAAGTTGGTGTGACTAGACGCGTTTATCACGCAGAAGGTTCTTTGAAACTTGGACTTGTTTGTGTGGT AGATAGTTTAGTTAGTAGCCGCGGCGTAGAGAATACGTTGCTGATGTTTTTATGGCCTGAAATTGGATGTGATGATCC GCTGAATTTAAGCATATAACTAAGCGGAAGAAAAGAAAATAACAATGATTCCCCT (828 bp) C.6.2.2 Dyabara ITS standard reference sequence (reference sequence GenBank serial number is EU185801, which starts in this gene Location is 1-104) GGACCGTGGCCTTTCCGATTCGTCGGCTTGGCCTAGGGAAGTCTTGTGAACCTTATCACTTAAAGGAAGGAGAAGT CGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTCACATTGAATACCCTTGCACAATAGTGCTCGGCTT CGGCATTTACGTTGTGTAAGCTTGCTTGCAGCTGTGACTCTACGTCATGGTCCACTTTTGGTGGTTTCGTATTTGTC GTTGCCMTGGCGACGTGGTTTCGGTCTTGTTCCGTTTCCATCCCTGCGCTTTTGCGTGGGACGTTGCCCCCTCCCAC CCCACCGGGTGTATGTTTACTGCGGTGTAACTACTGTAGTTGGCGTACACTTGGGTTATGCCGTGTCGATACTGATG TTACTAGTGATTGCTCTTTGAGTGGTTGTTAGTGCTACAGTGTTGTCACGGATGCTGCTCGTGGATCTAATAGATTC AAGCAGTTGCTGCTTCGTGCAGTGTTTTGCGTAGCGATTTCGTTACGATAATGCAACTCCGCTCGTTCATCGTCTTG CGTTGTTCGAGTTTGTTTAGAAATTATAAACTTTCAGCGATGGATGTCTTGGCTCACACAACGATGAAGGACGCAG CAAATTGCGATAAGCATTATGACTTGCAGACTTCTGCGATTTAACAGACCTCTGAACGTAACAAACACACCGCCTC TGCTCGCATGCGGTACTCCCGTTTCAGTGAGCCCCCTTTCCTAAAGGAACCACACTTTTACTCCTGGTAATAGTATG GTTAGTCCTTTGCGAGTGGGTGTTGTGACAATCACCTTAATTTCCATAGCATGCTMCCGGGTATCGCCACGTGTGA TCTCGAAGCTTCTTGTTGTAATTTATTACTCTAGGCTTCTTTTGAGATGTGCGGCTAGGAATTACTATTGCAGTATTT CTATAGCAAGTGGATGATGCTAGTGTTGTCAGTGCTATAAGTTTTGAATATCGCTCCTGAAATCGGGTGAGGCTAT CCGCTGAATTTAAGCATATAATTAAGCGGCAGAAAAGAAAATAACTAT (1044 bp) C.7 Babesia PCR electrophoresis results

Figure C.1 Results of 18S rRNA PCR electrophoresis of Babesia clinical samples (1 - positive control sample, 2 - 3 cases of Henan voles Babesia)

Samples, 4, 5 - Zhejiang, Yunnan voles Babesia case samples, 6, 7 - negative control and blank control samples)

Figure C.2 Results of ITS PCR electrophoresis of clinical samples of Babesia (1-positive control sample, 2, 3-sample of Babesia in Henan voles,

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