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Dockerfile

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+# 使用官方轻量级 Python 镜像
+FROM python:3.12-slim
+
+# 设置工作目录
+WORKDIR /app
+
+# 安装必要的系统库（针对 primer3 和 biopython 可能需要的依赖）
+RUN apt-get update && apt-get install -y \
+    build-essential \
+    && rm -rf /var/lib/apt/lists/*
+
+# 先复制 requirements.txt 以利用 Docker 缓存机制
+COPY requirements.txt .
+
+# 安装 Python 依赖
+RUN pip install --no-cache-dir -r requirements.txt
+
+# 复制项目所有文件到容器中
+COPY . .
+
+# 暴露 Flask 端口（需与 app.py 中的 port 一致）
+EXPOSE 5000
+
+# 启动程序
+# 注意：host 必须设为 0.0.0.0 才能让外部访问
+CMD ["python", "app.py"]

README.md

@@ -1,12 +1,108 @@
----
-title: FasterPrimer
-emoji: 📈
-colorFrom: purple
-colorTo: yellow
-sdk: docker
-pinned: false
-license: apache-2.0
-short_description: Fast & Hassle-Free RT-PCR Primer Design Website for Sybr Gre
----
-
-Check out the configuration reference at https://huggingface.co/docs/hub/spaces-config-reference
+---
+title: Primer Design Tool
+emoji: 🧬
+colorFrom: blue
+colorTo: green
+sdk: docker
+app_port: 5000
+pinned: false
+license: apache-2.0
+---
+# qPCR引物设计工具
+
+一个基于Web的qPCR引物设计工具，可以根据基因名称和物种自动设计跨越外显子交界点的引物序列。
+
+## 功能特点
+
+- 🧬 支持多种模式生物（人类、小鼠、大鼠、果蝇、线虫、斑马鱼）
+- 🎯 自动设计跨越外显子交界点的引物，避免基因组DNA扩增
+- 📊 提供详细的引物信息（Tm值、产物长度、序列等）
+- 💻 现代化的Web界面，支持移动端
+- ⚡ 基于NCBI数据库的实时查询
+- 📋 **批量处理功能** - 支持一次性处理多个基因
+- 📥 **多格式导出** - 支持Excel、CSV、JSON格式导出
+
+## 新增功能
+
+### 批量处理
+- 支持多种输入格式：每行一个基因、逗号分隔、空格分隔
+- 实时进度显示
+- 批量结果统计和汇总
+- 失败基因的详细错误信息
+
+### 导出功能
+- **Excel格式**: 完整的表格数据，包含所有引物信息
+- **CSV格式**: 兼容各种数据分析软件
+- **JSON格式**: 程序化处理的结构化数据
+- 自动生成带时间戳的文件名
+
+## 安装和运行
+
+### 1. 安装依赖
+
+```bash
+pip install -r requirements.txt
+```
+
+### 2. 运行应用
+
+```bash
+python app.py
+```
+
+### 3. 访问网页
+
+打开浏览器访问：http://localhost:5000
+
+## 使用方法
+
+### 单个基因设计
+1. 选择"单个基因"标签
+2. 输入基因名称（如：GAPDH, Gpr34）
+3. 选择目标物种
+4. 点击"设计引物"按钮
+5. 查看结果并可选择导出
+
+### 批量基因设计
+1. 选择"批量处理"标签
+2. 输入基因列表，支持多种格式：
+   - 每行一个：`GAPDH\nACTB\nTUBB3`
+   - 逗号分隔：`GAPDH, ACTB, TUBB3`
+   - 空格分隔：`GAPDH ACTB TUBB3`
+3. 选择目标物种
+4. 点击"批量设计引物"按钮
+5. 查看批量结果统计和详细信息
+6. 导出完整的批量结果
+
+## 导出格式说明
+
+### Excel格式 (.xlsx)
+包含完整的引物信息表格，字段包括：
+- 基因名称、RefSeq ID
+- 引物对编号、正反向引物序列
+- Tm值、产物长度
+- 外显子交界点信息、设计时间
+
+### CSV格式 (.csv)
+与Excel相同的数据，但以逗号分隔值格式保存，便于导入其他软件。
+
+### JSON格式 (.json)
+结构化的数据格式，包含：
+- 导出时间和统计信息
+- 完整的原始结果数据
+- 适合程序化处理
+
+## 技术栈
+
+- **后端**: Flask + Biopython + Primer3 + Pandas
+- **前端**: HTML5 + CSS3 + JavaScript
+- **数据源**: NCBI Entrez数据库
+- **导出**: openpyxl (Excel) + pandas (CSV/JSON)
+
+## 注意事项
+
+- 需要稳定的网络连接访问NCBI数据库
+- 批量处理时建议每次不超过20个基因，避免超时
+- 首次查询某个基因可能需要较长时间
+- 建议使用标准的基因符号进行查询
+- 导出的文件会自动添加时间戳避免重名

app.js

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+let currentResults = null;
+let currentBatchResults = null;
+
+// 标签页切换
+function switchTab(tabName) {
+    // 隐藏所有标签内容
+    document.querySelectorAll('.tab-content').forEach(tab => {
+        tab.classList.remove('active');
+    });
+    document.querySelectorAll('.tab-button').forEach(btn => {
+        btn.classList.remove('active');
+    });
+    
+    // 显示选中的标签
+    document.getElementById(tabName + '-tab').classList.add('active');
+    event.target.classList.add('active');
+    
+    // 隐藏结果区域
+    document.getElementById('results').style.display = 'none';
+    document.getElementById('batchResults').style.display = 'none';
+}
+
+// 单个基因设计
+document.getElementById('primerForm').addEventListener('submit', async function(e) {
+    e.preventDefault();
+    
+    const geneSymbol = document.getElementById('geneSymbol').value.trim();
+    const species = document.getElementById('species').value;
+    
+    if (!geneSymbol) {
+        alert('请输入基因名称');
+        return;
+    }
+    
+    // 显示加载状态
+    document.getElementById('loading').style.display = 'block';
+    document.getElementById('results').style.display = 'none';
+    document.getElementById('batchResults').style.display = 'none';
+    document.getElementById('submitBtn').disabled = true;
+    
+    try {
+        const response = await fetch('/design_primers', {
+            method: 'POST',
+            headers: {
+                'Content-Type': 'application/json',
+            },
+            body: JSON.stringify({
+                gene_symbol: geneSymbol,
+                species: species
+            })
+        });
+        
+        const data = await response.json();
+        
+        if (data.error) {
+            showError(data.error);
+        } else {
+            currentResults = [{ gene: geneSymbol, status: 'success', data: data }];
+            showResults(data);
+        }
+    } catch (error) {
+        showError('网络错误，请检查连接后重试');
+    } finally {
+        document.getElementById('loading').style.display = 'none';
+        document.getElementById('submitBtn').disabled = false;
+    }
+});
+
+// 批量基因设计
+document.getElementById('batchForm').addEventListener('submit', async function(e) {
+    e.preventDefault();
+    
+    const batchGenesText = document.getElementById('batchGenes').value.trim();
+    const species = document.getElementById('batchSpecies').value;
+    
+    if (!batchGenesText) {
+        alert('请输入基因列表');
+        return;
+    }
+    
+    // 解析基因列表
+    const geneList = parseGeneList(batchGenesText);
+    
+    if (geneList.length === 0) {
+        alert('未找到有效的基因名称');
+        return;
+    }
+    
+    // 显示进度条
+    document.getElementById('batchProgress').style.display = 'block';
+    document.getElementById('results').style.display = 'none';
+    document.getElementById('batchResults').style.display = 'none';
+    document.getElementById('batchSubmitBtn').disabled = true;
+    
+    try {
+        const response = await fetch('/batch_design_primers', {
+            method: 'POST',
+            headers: {
+                'Content-Type': 'application/json',
+            },
+            body: JSON.stringify({
+                gene_list: geneList,
+                species: species
+            })
+        });
+        
+        const data = await response.json();
+        currentBatchResults = data.results;
+        showBatchResults(data.results);
+        
+    } catch (error) {
+        showError('网络错误，请检查连接后重试');
+    } finally {
+        document.getElementById('batchProgress').style.display = 'none';
+        document.getElementById('batchSubmitBtn').disabled = false;
+    }
+});
+
+function parseGeneList(text) {
+    // 支持多种分隔符：换行、逗号、空格
+    return text
+        .split(/[\n,\s]+/)
+        .map(gene => gene.trim())
+        .filter(gene => gene.length > 0);
+}
+
+function showError(message) {
+    document.getElementById('results').innerHTML = `
+        <div class="error">
+            ❌ ${message}
+        </div>
+    `;
+    document.getElementById('results').style.display = 'block';
+}
+
+function showResults(data) {
+    const geneInfo = data.gene_info;
+    const primers = data.primers;
+    
+    // 显示基因信息
+    document.getElementById('geneInfo').innerHTML = `
+        <h3>📋 基因信息</h3>
+        <p><strong>基因名称:</strong> ${geneInfo.symbol}</p>
+        <p><strong>RefSeq ID:</strong> ${geneInfo.nm_id}</p>
+        <p><strong>外显子交界点:</strong> ${geneInfo.junctions.join(', ')}</p>
+    `;
+    
+    // 显示引物结果
+    let primerHtml = '<h3>🎯 推荐引物序列</h3>';
+    
+    primers.forEach(primer => {
+        primerHtml += `
+            <div class="primer-card">
+                <div class="primer-header">
+                    <span class="primer-id">引物对 ${primer.id}</span>
+                    <span class="product-size">${primer.product_size} bp</span>
+                </div>
+                
+                <div class="primer-sequences">
+                    <div class="sequence-box">
+                        <div class="sequence-label">正向引物 (Forward)</div>
+                        <div class="sequence">${primer.forward}</div>
+                    </div>
+                    <div class="sequence-box">
+                        <div class="sequence-label">反向引物 (Reverse)</div>
+                        <div class="sequence">${primer.reverse}</div>
+                    </div>
+                </div>
+                
+                <div class="tm-info">
+                    <span>正向Tm: ${primer.f_tm}°C</span>
+                    <span>反向Tm: ${primer.r_tm}°C</span>
+                    <span>${primer.junction_info}</span>
+                </div>
+            </div>
+        `;
+    });
+    
+    document.getElementById('primerResults').innerHTML = primerHtml;
+    document.getElementById('exportSection').style.display = 'block';
+    document.getElementById('results').style.display = 'block';
+}
+
+function showBatchResults(results) {
+    const successCount = results.filter(r => r.status === 'success').length;
+    const failedCount = results.filter(r => r.status === 'failed').length;
+    const totalPrimers = results
+        .filter(r => r.status === 'success')
+        .reduce((sum, r) => sum + r.data.primers.length, 0);
+    
+    // 显示统计信息
+    document.getElementById('resultSummary').innerHTML = `
+        <div class="summary-stats">
+            <div class="stat-item">
+                <div class="stat-number">${results.length}</div>
+                <div class="stat-label">总基因数</div>
+            </div>
+            <div class="stat-item">
+                <div class="stat-number">${successCount}</div>
+                <div class="stat-label">成功设计</div>
+            </div>
+            <div class="stat-item">
+                <div class="stat-number">${failedCount}</div>
+                <div class="stat-label">设计失败</div>
+            </div>
+            <div class="stat-item">
+                <div class="stat-number">${totalPrimers}</div>
+                <div class="stat-label">引物对总数</div>
+            </div>
+        </div>
+    `;
+    
+    // 显示失败的基因
+    const failedGenes = results.filter(r => r.status === 'failed');
+    if (failedGenes.length > 0) {
+        let failedHtml = '<div class="failed-genes"><h4>⚠️ 设计失败的基因</h4>';
+        failedGenes.forEach(gene => {
+            failedHtml += `
+                <div class="failed-gene">
+                    <strong>${gene.gene}:</strong> ${gene.error}
+                </div>
+            `;
+        });
+        failedHtml += '</div>';
+        document.getElementById('failedGenes').innerHTML = failedHtml;
+    } else {
+        document.getElementById('failedGenes').innerHTML = '';
+    }
+    
+    // 显示成功的引物结果
+    let primerHtml = '<h3>🎯 批量引物设计结果</h3>';
+    
+    results.filter(r => r.status === 'success').forEach(result => {
+        const geneInfo = result.data.gene_info;
+        const primers = result.data.primers;
+        
+        primerHtml += `<div style="margin-bottom: 40px; border: 2px solid #e1e5e9; border-radius: 8px; padding: 20px;">`;
+        primerHtml += `<h4 style="color: #4facfe; margin-bottom: 15px;">📋 ${geneInfo.symbol} (${geneInfo.nm_id})</h4>`;
+        
+        primers.forEach(primer => {
+            primerHtml += `
+                <div class="primer-card">
+                    <div class="primer-header">
+                        <span class="primer-id">引物对 ${primer.id}</span>
+                        <span class="product-size">${primer.product_size} bp</span>
+                    </div>
+                    
+                    <div class="primer-sequences">
+                        <div class="sequence-box">
+                            <div class="sequence-label">正向引物 (Forward)</div>
+                            <div class="sequence">${primer.forward}</div>
+                        </div>
+                        <div class="sequence-box">
+                            <div class="sequence-label">反向引物 (Reverse)</div>
+                            <div class="sequence">${primer.reverse}</div>
+                        </div>
+                    </div>
+                    
+                    <div class="tm-info">
+                        <span>正向Tm: ${primer.f_tm}°C</span>
+                        <span>反向Tm: ${primer.r_tm}°C</span>
+                        <span>${primer.junction_info}</span>
+                    </div>
+                </div>
+            `;
+        });
+        
+        primerHtml += '</div>';
+    });
+    
+    document.getElementById('batchPrimerResults').innerHTML = primerHtml;
+    document.getElementById('batchResults').style.display = 'block';
+}
+
+// 导出单个结果
+async function exportResults(format) {
+    if (!currentResults) {
+        alert('没有可导出的结果');
+        return;
+    }
+    
+    try {
+        const response = await fetch('/export_primers', {
+            method: 'POST',
+            headers: {
+                'Content-Type': 'application/json',
+            },
+            body: JSON.stringify({
+                format: format,
+                data: currentResults
+            })
+        });
+        
+        if (response.ok) {
+            const blob = await response.blob();
+            const url = window.URL.createObjectURL(blob);
+            const a = document.createElement('a');
+            a.href = url;
+            a.download = response.headers.get('Content-Disposition')?.split('filename=')[1] || `primers.${format}`;
+            document.body.appendChild(a);
+            a.click();
+            window.URL.revokeObjectURL(url);
+            document.body.removeChild(a);
+        } else {
+            alert('导出失败，请重试');
+        }
+    } catch (error) {
+        alert('导出失败：' + error.message);
+    }
+}
+
+// 导出批量结果
+async function exportBatchResults(format) {
+    if (!currentBatchResults) {
+        alert('没有可导出的结果');
+        return;
+    }
+    
+    try {
+        const response = await fetch('/export_primers', {
+            method: 'POST',
+            headers: {
+                'Content-Type': 'application/json',
+            },
+            body: JSON.stringify({
+                format: format,
+                data: currentBatchResults
+            })
+        });
+        
+        if (response.ok) {
+            const blob = await response.blob();
+            const url = window.URL.createObjectURL(blob);
+            const a = document.createElement('a');
+            a.href = url;
+            a.download = response.headers.get('Content-Disposition')?.split('filename=')[1] || `batch_primers.${format}`;
+            document.body.appendChild(a);
+            a.click();
+            window.URL.revokeObjectURL(url);
+            document.body.removeChild(a);
+        } else {
+            alert('导出失败，请重试');
+        }
+    } catch (error) {
+        alert('导出失败：' + error.message);
+    }
+}

app.py

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+from flask import Flask, render_template, request, jsonify, send_file
+from Bio import Entrez, SeqIO
+import primer3
+import ssl
+import pandas as pd
+import io
+import json
+from datetime import datetime
+
+app = Flask(__name__)
+ssl._create_default_https_context = ssl._create_unverified_context
+def get_ready_for_primers(gene_symbol, species="human"):
+    """获取基因序列和外显子交界点信息"""
+    Entrez.email = "your_email@example.com"
+    
+    # 1. 搜索基因并获取 NCBI 内部 ID
+    search_term = f"{gene_symbol}[Gene Name] AND {species}[Organism]"
+    handle = Entrez.esearch(db="gene", term=search_term)
+    record = Entrez.read(handle)
+    if not record["IdList"]: 
+        return {"error": "未找到该基因"}
+    gene_id = record["IdList"][0]
+
+    # 2. 获取该基因关联的 NM_ 编号
+    link_handle = Entrez.elink(dbfrom="gene", db="nucleotide", id=gene_id, term="srcdb_refseq[prop] AND mRNA[filter]")
+    link_record = Entrez.read(link_handle)
+    
+    try:
+        # 获取第一个关联的核苷酸 UID
+        nucl_id = link_record[0]["LinkSetDb"][0]["Link"][0]["Id"]
+        
+        # 3. 下载完整的 GenBank 格式数据
+        handle = Entrez.efetch(db="nucleotide", id=nucl_id, rettype="gb", retmode="text")
+        seq_record = SeqIO.read(handle, "genbank")
+        
+        # 提取外显子分界点
+        junctions = []
+        current_pos = 0
+        for feature in seq_record.features:
+            if feature.type == "exon":
+                start, end = feature.location.start, feature.location.end
+                current_pos += (end - start)
+                junctions.append(int(current_pos))
+        
+        if junctions: 
+            junctions.pop()  # 移除最后一个边界
+
+        return {
+            "symbol": gene_symbol,
+            "nm_id": seq_record.id,
+            "sequence": str(seq_record.seq),
+            "junctions": junctions
+        }
+    except Exception as e:
+        return {"error": f"获取基因信息失败: {str(e)}"}
+
+def design_qpcr_primers(gene_data):
+    """设计qPCR引物"""
+    if "error" in gene_data:
+        return gene_data
+    
+    seq_args = {
+        'SEQUENCE_ID': gene_data['nm_id'],
+        'SEQUENCE_TEMPLATE': gene_data['sequence'],
+        'SEQUENCE_OVERLAP_JUNCTION_LIST': gene_data['junctions'],
+    }
+
+    global_args = {
+        'PRIMER_OPT_SIZE': 20,
+        'PRIMER_MIN_SIZE': 18,
+        'PRIMER_MAX_SIZE': 25,
+        'PRIMER_OPT_TM': 60.0,
+        'PRIMER_MIN_TM': 57.0,
+        'PRIMER_MAX_TM': 63.0,
+        'PRIMER_TM_MAX_DIFF': 1.0,
+        'PRIMER_MIN_GC': 40.0,
+        'PRIMER_MAX_GC': 60.0,
+        'PRIMER_PRODUCT_SIZE_RANGE': [120, 300],
+        'PRIMER_NUM_RETURN': 5,
+    }
+
+    try:
+        results = primer3.bindings.design_primers(seq_args, global_args)
+        
+        primer_pairs = []
+        for i in range(global_args['PRIMER_NUM_RETURN']):
+            try:
+                pair = {
+                    "id": i + 1,
+                    "forward": results[f'PRIMER_LEFT_{i}_SEQUENCE'],
+                    "reverse": results[f'PRIMER_RIGHT_{i}_SEQUENCE'],
+                    "f_tm": f"{results[f'PRIMER_LEFT_{i}_TM']:.2f}",
+                    "r_tm": f"{results[f'PRIMER_RIGHT_{i}_TM']:.2f}",
+                    "product_size": results[f'PRIMER_PAIR_{i}_PRODUCT_SIZE'],
+                    "junction_info": "跨越外显子交界点"
+                }
+                primer_pairs.append(pair)
+            except KeyError:
+                break
+                
+        return {"primers": primer_pairs, "gene_info": gene_data}
+    except Exception as e:
+        return {"error": f"引物设计失败: {str(e)}"}
+
+@app.route('/')
+def index():
+    return render_template('index.html')
+
+@app.route('/design_primers', methods=['POST'])
+def design_primers_api():
+    data = request.json
+    gene_symbol = data.get('gene_symbol', '').strip()
+    species = data.get('species', 'human')
+    
+    if not gene_symbol:
+        return jsonify({"error": "请输入基因名称"})
+    
+    # 获取基因信息
+    gene_data = get_ready_for_primers(gene_symbol, species)
+    
+    # 设计引物
+    result = design_qpcr_primers(gene_data)
+    
+    return jsonify(result)
+
+@app.route('/batch_design_primers', methods=['POST'])
+def batch_design_primers_api():
+    data = request.json
+    gene_list = data.get('gene_list', [])
+    species = data.get('species', 'human')
+    
+    if not gene_list:
+        return jsonify({"error": "请输入基因列表"})
+    
+    results = []
+    for gene_symbol in gene_list:
+        gene_symbol = gene_symbol.strip()
+        if not gene_symbol:
+            continue
+            
+        # 获取基因信息
+        gene_data = get_ready_for_primers(gene_symbol, species)
+        
+        # 设计引物
+        result = design_qpcr_primers(gene_data)
+        
+        if "error" in result:
+            results.append({
+                "gene": gene_symbol,
+                "status": "failed",
+                "error": result["error"]
+            })
+        else:
+            results.append({
+                "gene": gene_symbol,
+                "status": "success",
+                "data": result
+            })
+    
+    return jsonify({"results": results})
+
+@app.route('/export_primers', methods=['POST'])
+def export_primers():
+    data = request.json
+    export_format = data.get('format', 'excel')  # excel, csv, json
+    results_data = data.get('data', [])
+    
+    if export_format == 'excel':
+        return export_to_excel(results_data)
+    elif export_format == 'csv':
+        return export_to_csv(results_data)
+    elif export_format == 'json':
+        return export_to_json(results_data)
+    else:
+        return jsonify({"error": "不支持的导出格式"})
+
+def export_to_excel(results_data):
+    """导出为Excel格式"""
+    rows = []
+    
+    for result in results_data:
+        if result.get('status') == 'success':
+            gene_info = result['data']['gene_info']
+            primers = result['data']['primers']
+            
+            for primer in primers:
+                rows.append({
+                    '基因名称': gene_info['symbol'],
+                    'RefSeq ID': gene_info['nm_id'],
+                    '引物对编号': primer['id'],
+                    '正向引物序列': primer['forward'],
+                    '反向引物序列': primer['reverse'],
+                    '正向引物Tm(°C)': primer['f_tm'],
+                    '反向引物Tm(°C)': primer['r_tm'],
+                    '产物长度(bp)': primer['product_size'],
+                    '外显子交界点': ', '.join(map(str, gene_info['junctions'])),
+                    '设计时间': datetime.now().strftime('%Y-%m-%d %H:%M:%S')
+                })
+        else:
+            rows.append({
+                '基因名称': result['gene'],
+                'RefSeq ID': 'N/A',
+                '引物对编号': 'N/A',
+                '正向引物序列': 'N/A',
+                '反向引物序列': 'N/A',
+                '正向引物Tm(°C)': 'N/A',
+                '反向引物Tm(°C)': 'N/A',
+                '产物长度(bp)': 'N/A',
+                '外显子交界点': 'N/A',
+                '设计时间': datetime.now().strftime('%Y-%m-%d %H:%M:%S'),
+                '错误信息': result.get('error', '未知错误')
+            })
+    
+    df = pd.DataFrame(rows)
+    
+    # 创建Excel文件
+    output = io.BytesIO()
+    with pd.ExcelWriter(output, engine='openpyxl') as writer:
+        df.to_excel(writer, sheet_name='引物设计结果', index=False)
+    
+    output.seek(0)
+    filename = f"qPCR_primers_{datetime.now().strftime('%Y%m%d_%H%M%S')}.xlsx"
+    
+    return send_file(
+        output,
+        mimetype='application/vnd.openxmlformats-officedocument.spreadsheetml.sheet',
+        as_attachment=True,
+        download_name=filename
+    )
+
+def export_to_csv(results_data):
+    """导出为CSV格式"""
+    rows = []
+    
+    for result in results_data:
+        if result.get('status') == 'success':
+            gene_info = result['data']['gene_info']
+            primers = result['data']['primers']
+            
+            for primer in primers:
+                rows.append({
+                    '基因名称': gene_info['symbol'],
+                    'RefSeq ID': gene_info['nm_id'],
+                    '引物对编号': primer['id'],
+                    '正向引物序列': primer['forward'],
+                    '反向引物序列': primer['reverse'],
+                    '正向引物Tm(°C)': primer['f_tm'],
+                    '反向引物Tm(°C)': primer['r_tm'],
+                    '产物长度(bp)': primer['product_size'],
+                    '外显子交界点': ', '.join(map(str, gene_info['junctions'])),
+                    '设计时间': datetime.now().strftime('%Y-%m-%d %H:%M:%S')
+                })
+    
+    df = pd.DataFrame(rows)
+    
+    output = io.StringIO()
+    df.to_csv(output, index=False, encoding='utf-8-sig')
+    
+    filename = f"qPCR_primers_{datetime.now().strftime('%Y%m%d_%H%M%S')}.csv"
+    
+    return send_file(
+        io.BytesIO(output.getvalue().encode('utf-8-sig')),
+        mimetype='text/csv',
+        as_attachment=True,
+        download_name=filename
+    )
+
+def export_to_json(results_data):
+    """导出为JSON格式"""
+    export_data = {
+        "export_time": datetime.now().strftime('%Y-%m-%d %H:%M:%S'),
+        "total_genes": len(results_data),
+        "results": results_data
+    }
+    
+    filename = f"qPCR_primers_{datetime.now().strftime('%Y%m%d_%H%M%S')}.json"
+    
+    return send_file(
+        io.BytesIO(json.dumps(export_data, ensure_ascii=False, indent=2).encode('utf-8')),
+        mimetype='application/json',
+        as_attachment=True,
+        download_name=filename
+    )
+
+if __name__ == '__main__':
+    app.run(debug=True, host='0.0.0.0', port=5000)

index.html

@@ -0,0 +1,536 @@
+<!DOCTYPE html>
+<html lang="zh-CN">
+<head>
+    <meta charset="UTF-8">
+    <meta name="viewport" content="width=device-width, initial-scale=1.0">
+    <title>qPCR引物设计工具</title>
+    <style>
+        * {
+            margin: 0;
+            padding: 0;
+            box-sizing: border-box;
+        }
+        
+        body {
+            font-family: 'Segoe UI', Tahoma, Geneva, Verdana, sans-serif;
+            background: linear-gradient(135deg, #667eea 0%, #764ba2 100%);
+            min-height: 100vh;
+            padding: 20px;
+        }
+        
+        .container {
+            max-width: 1200px;
+            margin: 0 auto;
+            background: white;
+            border-radius: 15px;
+            box-shadow: 0 20px 40px rgba(0,0,0,0.1);
+            overflow: hidden;
+        }
+        
+        .header {
+            background: linear-gradient(135deg, #4facfe 0%, #00f2fe 100%);
+            color: white;
+            padding: 30px;
+            text-align: center;
+        }
+        
+        .header h1 {
+            font-size: 2.5em;
+            margin-bottom: 10px;
+        }
+        
+        .header p {
+            font-size: 1.1em;
+            opacity: 0.9;
+        }
+        
+        .form-section {
+            padding: 40px;
+        }
+        
+        .tab-container {
+            margin-bottom: 30px;
+        }
+        
+        .tab-buttons {
+            display: flex;
+            border-bottom: 2px solid #e1e5e9;
+            margin-bottom: 20px;
+        }
+        
+        .tab-button {
+            padding: 12px 24px;
+            background: none;
+            border: none;
+            cursor: pointer;
+            font-size: 16px;
+            font-weight: 600;
+            color: #666;
+            border-bottom: 3px solid transparent;
+            transition: all 0.3s;
+        }
+        
+        .tab-button.active {
+            color: #4facfe;
+            border-bottom-color: #4facfe;
+        }
+        
+        .tab-content {
+            display: none;
+        }
+        
+        .tab-content.active {
+            display: block;
+        }
+        
+        .batch-input {
+            margin-bottom: 20px;
+        }
+        
+        .batch-textarea {
+            width: 100%;
+            min-height: 150px;
+            padding: 15px;
+            border: 2px solid #e1e5e9;
+            border-radius: 8px;
+            font-size: 14px;
+            font-family: 'Courier New', monospace;
+            resize: vertical;
+        }
+        
+        .batch-help {
+            background: #f8f9fa;
+            padding: 15px;
+            border-radius: 8px;
+            margin-bottom: 20px;
+            font-size: 14px;
+            color: #666;
+        }
+        
+        .export-section {
+            background: #f8f9fa;
+            padding: 20px;
+            border-radius: 8px;
+            margin-top: 20px;
+        }
+        
+        .export-buttons {
+            display: flex;
+            gap: 10px;
+            flex-wrap: wrap;
+        }
+        
+        .export-btn {
+            background: #28a745;
+            color: white;
+            padding: 10px 20px;
+            border: none;
+            border-radius: 6px;
+            cursor: pointer;
+            font-size: 14px;
+            font-weight: 600;
+            transition: background 0.3s;
+        }
+        
+        .export-btn:hover {
+            background: #218838;
+        }
+        
+        .export-btn:disabled {
+            background: #6c757d;
+            cursor: not-allowed;
+        }
+        
+        .batch-progress {
+            display: none;
+            margin: 20px 0;
+        }
+        
+        .progress-bar {
+            width: 100%;
+            height: 20px;
+            background: #e1e5e9;
+            border-radius: 10px;
+            overflow: hidden;
+        }
+        
+        .progress-fill {
+            height: 100%;
+            background: linear-gradient(90deg, #4facfe, #00f2fe);
+            width: 0%;
+            transition: width 0.3s;
+        }
+        
+        .progress-text {
+            text-align: center;
+            margin-top: 10px;
+            font-weight: 600;
+        }
+        
+        .batch-results {
+            display: none;
+        }
+        
+        .result-summary {
+            background: white;
+            padding: 20px;
+            border-radius: 8px;
+            margin-bottom: 20px;
+            display: flex;
+            justify-content: space-between;
+            align-items: center;
+        }
+        
+        .summary-stats {
+            display: flex;
+            gap: 30px;
+        }
+        
+        .stat-item {
+            text-align: center;
+        }
+        
+        .stat-number {
+            font-size: 24px;
+            font-weight: bold;
+            color: #4facfe;
+        }
+        
+        .stat-label {
+            font-size: 12px;
+            color: #666;
+            text-transform: uppercase;
+        }
+        
+        .failed-genes {
+            background: #fff3cd;
+            border: 1px solid #ffeaa7;
+            padding: 15px;
+            border-radius: 8px;
+            margin-bottom: 20px;
+        }
+        
+        .failed-gene {
+            margin-bottom: 10px;
+            padding: 8px;
+            background: white;
+            border-radius: 4px;
+            border-left: 3px solid #e17055;
+        }
+        
+        .form-group {
+            margin-bottom: 25px;
+        }
+        
+        label {
+            display: block;
+            margin-bottom: 8px;
+            font-weight: 600;
+            color: #333;
+        }
+        
+        input, select {
+            width: 100%;
+            padding: 12px 15px;
+            border: 2px solid #e1e5e9;
+            border-radius: 8px;
+            font-size: 16px;
+            transition: border-color 0.3s;
+        }
+        
+        input:focus, select:focus {
+            outline: none;
+            border-color: #4facfe;
+        }
+        
+        .btn {
+            background: linear-gradient(135deg, #667eea 0%, #764ba2 100%);
+            color: white;
+            padding: 15px 30px;
+            border: none;
+            border-radius: 8px;
+            font-size: 16px;
+            font-weight: 600;
+            cursor: pointer;
+            transition: transform 0.2s;
+            width: 100%;
+        }
+        
+        .btn:hover {
+            transform: translateY(-2px);
+        }
+        
+        .btn:disabled {
+            opacity: 0.6;
+            cursor: not-allowed;
+            transform: none;
+        }
+        
+        .loading {
+            display: none;
+            text-align: center;
+            padding: 20px;
+        }
+        
+        .spinner {
+            border: 4px solid #f3f3f3;
+            border-top: 4px solid #4facfe;
+            border-radius: 50%;
+            width: 40px;
+            height: 40px;
+            animation: spin 1s linear infinite;
+            margin: 0 auto 10px;
+        }
+        
+        @keyframes spin {
+            0% { transform: rotate(0deg); }
+            100% { transform: rotate(360deg); }
+        }
+        
+        .results {
+            display: none;
+            padding: 40px;
+            background: #f8f9fa;
+        }
+        
+        .gene-info {
+            background: white;
+            padding: 20px;
+            border-radius: 8px;
+            margin-bottom: 30px;
+            border-left: 4px solid #4facfe;
+        }
+        
+        .primer-card {
+            background: white;
+            border-radius: 8px;
+            padding: 20px;
+            margin-bottom: 20px;
+            box-shadow: 0 2px 10px rgba(0,0,0,0.1);
+        }
+        
+        .primer-header {
+            display: flex;
+            justify-content: space-between;
+            align-items: center;
+            margin-bottom: 15px;
+        }
+        
+        .primer-id {
+            background: #4facfe;
+            color: white;
+            padding: 5px 15px;
+            border-radius: 20px;
+            font-weight: 600;
+        }
+        
+        .product-size {
+            background: #e8f5e8;
+            color: #2d5a2d;
+            padding: 5px 15px;
+            border-radius: 20px;
+            font-weight: 600;
+        }
+        
+        .primer-sequences {
+            display: grid;
+            grid-template-columns: 1fr 1fr;
+            gap: 20px;
+            margin-bottom: 15px;
+        }
+        
+        .sequence-box {
+            background: #f8f9fa;
+            padding: 15px;
+            border-radius: 6px;
+            border-left: 3px solid #4facfe;
+        }
+        
+        .sequence-label {
+            font-weight: 600;
+            color: #666;
+            margin-bottom: 5px;
+        }
+        
+        .sequence {
+            font-family: 'Courier New', monospace;
+            font-size: 14px;
+            word-break: break-all;
+            background: white;
+            padding: 8px;
+            border-radius: 4px;
+            border: 1px solid #e1e5e9;
+        }
+        
+        .tm-info {
+            display: flex;
+            justify-content: space-between;
+            font-size: 14px;
+            color: #666;
+        }
+        
+        .error {
+            background: #fee;
+            color: #c33;
+            padding: 15px;
+            border-radius: 8px;
+            border-left: 4px solid #c33;
+            margin: 20px 0;
+        }
+        
+        @media (max-width: 768px) {
+            .primer-sequences {
+                grid-template-columns: 1fr;
+            }
+            
+            .primer-header {
+                flex-direction: column;
+                gap: 10px;
+            }
+        }
+    </style>
+</head>
+<body>
+    <div class="container">
+        <div class="header">
+            <h1>🧬 qPCR引物设计工具</h1>
+            <p>输入基因名称和物种，自动设计跨越外显子交界点的qPCR引物</p>
+            <p style="margin-top: 15px; font-size: 0.9em; opacity: 0.8;">Developed by PharC | Contact: pc794797023@163.com</p>
+        </div>
+        
+        <div class="form-section">
+            <div class="tab-container">
+                <div class="tab-buttons">
+                    <button type="button" class="tab-button active" onclick="switchTab('single')">
+                        🧬 单个基因
+                    </button>
+                    <button type="button" class="tab-button" onclick="switchTab('batch')">
+                        📋 批量处理
+                    </button>
+                </div>
+                
+                <!-- 单个基因设计 -->
+                <div id="single-tab" class="tab-content active">
+                    <form id="primerForm">
+                        <div class="form-group">
+                            <label for="geneSymbol">基因名称 *</label>
+                            <input type="text" id="geneSymbol" name="geneSymbol" placeholder="例如: GAPDH, Gpr34" required>
+                        </div>
+                        
+                        <div class="form-group">
+                            <label for="species">物种</label>
+                            <select id="species" name="species">
+                                <option value="human">人类 (Human)</option>
+                                <option value="Mus musculus">小鼠 (Mouse)</option>
+                                <option value="Rattus norvegicus">大鼠 (Rat)</option>
+                                <option value="Drosophila melanogaster">果蝇 (Drosophila)</option>
+                                <option value="Caenorhabditis elegans">线虫 (C. elegans)</option>
+                                <option value="Danio rerio">斑马鱼 (Zebrafish)</option>
+                            </select>
+                        </div>
+                        
+                        <button type="submit" class="btn" id="submitBtn">
+                            🔬 设计引物
+                        </button>
+                    </form>
+                </div>
+                
+                <!-- 批量基因设计 -->
+                <div id="batch-tab" class="tab-content">
+                    <form id="batchForm">
+                        <div class="batch-help">
+                            💡 <strong>使用说明：</strong>每行输入一个基因名称，支持多种格式：
+                            <br>• 一行一个基因：GAPDH
+                            <br>• 逗号分隔：GAPDH, ACTB, TUBB3
+                            <br>• 空格分隔：GAPDH ACTB TUBB3
+                        </div>
+                        
+                        <div class="form-group">
+                            <label for="batchGenes">基因列表 *</label>
+                            <textarea 
+                                id="batchGenes" 
+                                class="batch-textarea" 
+                                placeholder="请输入基因名称，每行一个或用逗号/空格分隔：&#10;GAPDH&#10;ACTB&#10;TUBB3&#10;或者：GAPDH, ACTB, TUBB3"
+                                required
+                            ></textarea>
+                        </div>
+                        
+                        <div class="form-group">
+                            <label for="batchSpecies">物种</label>
+                            <select id="batchSpecies" name="batchSpecies">
+                                <option value="human">人类 (Human)</option>
+                                <option value="Mus musculus">小鼠 (Mouse)</option>
+                                <option value="Rattus norvegicus">大鼠 (Rat)</option>
+                                <option value="Drosophila melanogaster">果蝇 (Drosophila)</option>
+                                <option value="Caenorhabditis elegans">线虫 (C. elegans)</option>
+                                <option value="Danio rerio">斑马鱼 (Zebrafish)</option>
+                            </select>
+                        </div>
+                        
+                        <button type="submit" class="btn" id="batchSubmitBtn">
+                            🚀 批量设计引物
+                        </button>
+                    </form>
+                    
+                    <div class="batch-progress" id="batchProgress">
+                        <div class="progress-bar">
+                            <div class="progress-fill" id="progressFill"></div>
+                        </div>
+                        <div class="progress-text" id="progressText">正在处理: 0/0</div>
+                    </div>
+                </div>
+            </div>
+        </div>
+        
+        <div class="loading" id="loading">
+            <div class="spinner"></div>
+            <p>正在设计引物，请稍候...</p>
+        </div>
+        
+        <div class="results" id="results">
+            <div class="gene-info" id="geneInfo"></div>
+            <div id="primerResults"></div>
+            
+            <div class="export-section" id="exportSection" style="display: none;">
+                <h4>📥 导出结果</h4>
+                <p>选择导出格式：</p>
+                <div class="export-buttons">
+                    <button class="export-btn" onclick="exportResults('excel')">
+                        📊 Excel格式
+                    </button>
+                    <button class="export-btn" onclick="exportResults('csv')">
+                        📄 CSV格式
+                    </button>
+                    <button class="export-btn" onclick="exportResults('json')">
+                        🔧 JSON格式
+                    </button>
+                </div>
+            </div>
+        </div>
+        
+        <div class="batch-results" id="batchResults">
+            <div class="result-summary" id="resultSummary"></div>
+            <div id="failedGenes"></div>
+            <div id="batchPrimerResults"></div>
+            
+            <div class="export-section">
+                <h4>📥 导出批量结果</h4>
+                <p>选择导出格式：</p>
+                <div class="export-buttons">
+                    <button class="export-btn" onclick="exportBatchResults('excel')">
+                        📊 Excel格式
+                    </button>
+                    <button class="export-btn" onclick="exportBatchResults('csv')">
+                        📄 CSV格式
+                    </button>
+                    <button class="export-btn" onclick="exportBatchResults('json')">
+                        🔧 JSON格式
+                    </button>
+                </div>
+            </div>
+        </div>
+    </div>
+
+    <script src="{{ url_for('static', filename='app.js') }}"></script>
+</body>
+</html>

requirements.txt

@@ -0,0 +1,5 @@
+Flask
+biopython
+primer3-py
+pandas
+openpyxl