多重實時熒光定量PCR目標確立

建立可同時鑒別DPCL、DPCC、DPCE和DPCN四種真菌的多重實時熒光定量PCR梵音檢測體系

2

引物（和）探針組設(shè)計和評估

基于TEF基因設(shè)計4種菌種特異性引物探針組：

1) sequence alignments：ClustalW as implemented in BioEdit  (version 7.1.3.0);

2) Tm and potential secondary structures of primers and probes  evaluated with Gene Runner (Version 6.5.52   Beta);

3) specificity of the primers evaluated by NCBI’s Primer-BLAST  (https://www.ncbi.nlm.nih.gov/tools/primer-blast/);

1）多重實時熒光PCR的正反向引物、熒光標記探針3個寡核苷酸均須是序列高度特異（如與其他序列結(jié)合會降低PCR效率、出現(xiàn)假陽性結(jié)果）；

2）BLAST軟件序列特異性檢查；

3）4重實時熒光PCR體系需鑒定12個寡核苷酸序列的特異性。

4種真菌TEF序列通用正、反向引物設(shè)計

（擴增4種病原菌的TEF基因片段）

擴增產(chǎn)物既作熒光定量PCR引物探針特異性、靈敏度驗證測試靶，又用作單重、多重定量PCR反應(yīng)標準曲線內(nèi)參標準品

大豆看家基因GmUKN2引物設(shè)計

（定量樣品中大豆DNA數(shù)量）

大豆DNA作為外參，計算單位質(zhì)量的豆種中病原菌DNA量，評估真菌感染程度

待測和各種對照品DNA提取

PCR產(chǎn)物制作定量內(nèi)參標準品和實時熒光定量PCR檢測目標

其他非目標病原菌DNA的分別提取

設(shè)立單獨反應(yīng)孔作對照，評估各引物-探針組的特異性

健康大豆植株中提取DNA

作陰性對照設(shè)立單獨反應(yīng)孔，驗證探針的特異性）

人工DPCL菌屬感染的大豆莖稈、種子和種皮中分別提取DNA

用于實際檢驗四重?zé)晒舛縋CR檢測方法的有效性

3-2

4種目標菌種TEF DNA序列PCR擴增

Diaporthe isolates TEF regions  amplifications

TEF regions primers EF1-728F (5’-CATCGAGAAGTTCGAGAAGG-3’) and EF1-986R  (5’-TACTTGAAGGAACCCTTACC-3’) in individual reactions for the different  Diaporthe isolates.

25μl Reaction mixtures: 2.5μl 10x Taq buffer with (NH4)2SO4 (Thermo  Fisher Scientific), 2.5μl 2 mM dNTPs, 2.5μl MgCl2 (25 mM), 12.5 pmol of each  primer set, 1μl Taq DNA polymerase (1 U/μl), and 1μl genomic DNA.

Amplification conditions: 3min - 95℃, 35 cycles  (denaturation 30s -95℃, annealing 30s  - 68℃, elongation 30s - 72℃), and a final 5min -72℃ elongation.

PCR products were checked on 2%   agarose gels electrophoresis.

PCR products were purified using the  PEQGOLD Cycle-Pure Kit (PEQLAB Biotechnologie) and determined by Qubit 2.0 Fluorometer (Thermo Fisher Scientific).

PCR products containing 109  to 104 copies/μl, diluted with DNA prepared from soybean tissue, roughly  20ng, 2ng, 200pg, 20pg, 2pg, 0.2pg, and 0.02pg DNA per reaction.

1）本例內(nèi)參標準品用的是各目標菌屬的TEF基因擴增片段；

2）文中實驗結(jié)果中的標準品起始濃度范圍為20ng – 0.02pg共7個數(shù)量級；實際配制了覆蓋10-1010 copies共10個數(shù)量級的標準品。

3-3

4種目標真菌基因組DNA樣品制備測定

genomic DNA of the Diaporthe isolates  was both  determined by measuring absorption at 260nm and by Qubit 2.0.

1:10–1:106 dilution  series were prepared with 50 μg/ml DNA prepared from healthy soybean  tissue.

1）各目標菌種DNA提取樣品用健康大豆DNA稀釋液稀釋成1/10、1/100、1/1000三個濃度梯度（模擬真實檢測樣品基質(zhì)條件測試），以降低DNA樣品中PCR抑制成分干擾，確認實測條件。

2）目標菌株DNA樣品用于評估多重實時熒光定量PCR體系引物-探針組的特異性。

4-1

Assessing the specificity of the primer-probe sets

singleplex real-time PCR assays

amplification efficiency Assessing: using serial dilutions of PCR products(10  to 109 copies) for amplification standard curves;

Assessing conditions of the test for Diaporthe infestations,  1:1,1:10,1:100,1:1000 serial dilutions of genomic DNAs from the different  Diaporthe isolates were also used for quantification standard curves;

non-target Diaporthe species

No-template controls were included

20μl Reaction mixtures:10μl ready to use SensiFAST Probe No-ROX mix  (2x) (Bioline GmbH),8 pmol of each  forward and reverse primers,2 pmol probe,2μl template DNA；

Amplification conditions: 3 min at 95℃ and 40  cycles of 95℃ - 15s and 60℃ - 45s；

CFX96 Real-Time PCR system (Bio-Rad)

FrameStar 96-Well Skirted PCR Plates (4titude, USA)

DPCL、DPCC、DPCE和DPCN四引物-探針組擴增效率、特異性單重實時熒光定量反應(yīng)驗證

1）分別用四種菌株DNA、非目標菌株DNA及空白對照，逐一測試每各引物-探針組特異性；

2）以四TEF基因PCR產(chǎn)物作標準品分別建立單重?zé)晒舛繕藴是€，檢驗在避免PCR抑制干擾理想情況下單重反應(yīng)的擴增效率和檢測限；

3）用4種菌株DNA提取液的1:1、1:10、1:100、1:1000稀釋液為內(nèi)參標準品構(gòu)建標準曲線，評價實測條件下的擴增效率和條件；

4-2

Assessing the specificity of the primer-probe sets

Duplex real-time PCR assays with  primer-probe combinations

（consists of 4 in individual reactions: parallel dilution series of both species，and one undiluted (20ng) genomic DNA combined with  1:1000 diluted genomic DNA of the other species）

DPCL+DPCC

DPCL+DPCE

DPCL+DPCN

DPCC+DPCE

DPCC+DPCN

DPCE+DPCN

Reaction mixtures：10μl 2x SensiFAST Probe No-ROX mix,  and a reduced amount of 4pmol of each primers set, 1 pmol of each of the two  probes, and 2μl template DNA.

Amplification conditions: as Duplex

引物探針組特異性的交叉檢驗：將4個引物探針組兩兩配對（6種組合）進行雙重?zé)晒釶CR實驗。

1）對平行稀釋DNA混合測試：分別對兩種病原菌的未稀釋(20ng)的DNA混合物、兩種1/1000稀釋DNA混合物檢測；

2）對兩種不同稀釋度真菌DNA（一種1:1而另一種1/1000稀釋）的混合測試，檢驗當(dāng)一種模板濃度遠高于另一個模板濃度情況下競爭抑制和多重反應(yīng)有效性。如對DPCL+DPCC組合的測試分為：

①未稀釋DPCL+稀釋DPCC DNA混合；

②稀釋DPCL+未稀釋DPCC DNA混合；

4-3

Specificity of the quadruplex real-time PCR assay

1）4種引物-探針組組合同時對2種真菌檢測

DPCL + DPCC；DPCL + DPCE；DPCL + DPCN；DPCC + DPCE；DPCC + DPCN；DPCE + DPCN

2）4種引物-探針組組合同時對檢測3種真菌檢測

DPCL + DPCC + DPCE；DPCL + DPCC + DPCN；DPCL + DPCE +  DPCN；DPCC + DPCE + DPCN

3）4種引物-探針組組合同時對4種真菌檢測

DPCL + DPCC + DPCE + DPCN

4）4種引物-探針組組合對其他大豆病原菌種檢測

Reaction mixtures：as Duplex

Amplification conditions: as singleplex.

四重實時熒光定量PCR檢測體系特異性檢驗

1）4個引物探針組混合，測試2種、3種靶DNA共存時的特異性。

2）4個引物探針組能同時檢測四種靶DNA的特異性。

3）所有情況下，各引物探針組均專一性擴增靶DNA。非目標菌體、健康大豆葉和莖的DNA測試均無擴增。

4-4

多重?zé)晒舛縋CR反應(yīng)體系標準曲線的建立

Standard curves establishing for the quadruplex real-time PCR assay

DNA isolates from 4 target Diaporthe species were diluted with DNA  prepared from soybean tissue, roughly 20ng, 2ng, 200pg, 20pg, 2pg, 0.2pg, and  0.02pg DNA per reaction；

The standard curves used to calculate the amount of Diaporthe DNA in  ng per reaction；

a rough estimate of the limit of detection (LOD) and a Cq cut-off  values.

分別取4種菌株DNA，用健康大豆DNA連續(xù)稀釋制備的內(nèi)參標準品進行標準品標準曲線實驗。

分別建立4個靶基因標準曲線；

確定各靶標檢測的LOD（和Cq的cut-off值）

5

多重?zé)晒舛縋CR反應(yīng)方法的實測驗證

Validation of the quadruplex real-time PCR assay

1）Infected soybean stems

detect DNA from Diaporthe spp. prepared from stem samples covered with  pycnidia.

D. longicolla DNA was detected in all tested samples with visible  symptoms.

For healthy stem samples the primer-probe sets produced no  amplification.

2）Screening soybean seeds

All four Diaporthe species could be detected in different seed samples  known to contain seeds infected with Diaporthe strains.

For healthy seeds no amplification.

用實際感染目標真菌的大豆植株樣品和大豆種子和健康大豆種對照，檢驗多重實時熒光定量PCR檢測體系有效性。

6

Reaction mixtures for quantifying soybean DNA

Reaction mixtures：10μl 2x SensiFAST SYBR No-ROX mix, 8  pmol each of primers GmUKN2f and GmUKN2r, and 2μl template DNA.

as Duplex

Amplification conditions: as singleplex.

No-template controls were included

用大豆GmUKN2基因作為外參，計算單位大豆中真菌基因含量，評估感染風(fēng)險和鑒定豆種品質(zhì)。